lipofectamine 2000  (Thermo Fisher)


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    Name:
    Lipofectamine 2000 Transfection Reagent
    Description:
    Lipofectamine 2000 Transfection Reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines Researchers use Lipofectamine 2000 Reagent for siRNA and shRNA based gene knockdown experiments as well as for gene expression studies With Lipofectamine 2000 Transfection Reagent you ll get • Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression see table • Superior performance for co transfection of siRNA and plasmid DNA• Proven efficacy in the presence of serum eliminates the need to change media following transfection• Reliable performance for high throughput applications• The best choice for establishing stable cell linesA high performance transfection reagent for gene expression and gene silencingLipofectamine 2000 Transfection Reagent works effectively with all common cell lines as well as with many challenging ones and can be used in media with or without serum For gene silencing Lipofectamine 2000 Transfection Reagent s high efficiency transfections provide the high levels of gene knockdown needed to produce convincing results Lipofectamine 2000 Transfection Reagent is the number one choice for co transfection given its effectiveness for transfecting both siRNA and plasmid DNA Lipofectamine 2000 Transfection Reagent is easy to use simply mix with nucleic acid and add to cell culture Ideal for high throughput workSimplicity and speed combined with high transfection efficiency make Lipofectamine 2000 Transfection Reagent ideal for transient protein expression or high throughput RNAi transfections Transfection conditions can be easily established for automated or robotic systems used in such applications
    Catalog Number:
    11668019
    Price:
    None
    Applications:
    Cell Culture|Plasmid Transfection|RNAi Transfection|Stem Cell & Primary Cell Transfections|Synthetic siRNA Transfection|shRNA & miR RNAi Plasmid Transfection|Transfection
    Category:
    Cell Culture Transfection Reagents
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    Structured Review

    Thermo Fisher lipofectamine 2000
    Transfection efficiency and stability of the mimics or inhibitor and effects of miR-140-5p in A549 cells on the Smad/TGF-β1 and Wnt1/β-catenin signaling pathways. miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC were individually mixed with <t>Lipofectamine</t> 2000 (Invitrogen, Carlsbad, CA, USA) and placed into the cell culture to transfect A549 cells. After 48 h of transfection, (a) fluorescence expression of transfected cells after miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC transfection (×50); (b) transfection efficiency of miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC; (c) and (d) relative miR-140-5p expression levels were determined by qRT-PCR; (e)–(h) Wnt1, β-catenin, p-Smad3, and TGF-β1 expression levels were measured by Western blot analysis. Data from three independent experiments are presented as mean ± SD. * P
    Lipofectamine 2000 Transfection Reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines Researchers use Lipofectamine 2000 Reagent for siRNA and shRNA based gene knockdown experiments as well as for gene expression studies With Lipofectamine 2000 Transfection Reagent you ll get • Exceptional transfection efficiency in the broadest range of cell lines and the highest levels of recombinant protein expression see table • Superior performance for co transfection of siRNA and plasmid DNA• Proven efficacy in the presence of serum eliminates the need to change media following transfection• Reliable performance for high throughput applications• The best choice for establishing stable cell linesA high performance transfection reagent for gene expression and gene silencingLipofectamine 2000 Transfection Reagent works effectively with all common cell lines as well as with many challenging ones and can be used in media with or without serum For gene silencing Lipofectamine 2000 Transfection Reagent s high efficiency transfections provide the high levels of gene knockdown needed to produce convincing results Lipofectamine 2000 Transfection Reagent is the number one choice for co transfection given its effectiveness for transfecting both siRNA and plasmid DNA Lipofectamine 2000 Transfection Reagent is easy to use simply mix with nucleic acid and add to cell culture Ideal for high throughput workSimplicity and speed combined with high transfection efficiency make Lipofectamine 2000 Transfection Reagent ideal for transient protein expression or high throughput RNAi transfections Transfection conditions can be easily established for automated or robotic systems used in such applications
    https://www.bioz.com/result/lipofectamine 2000/product/Thermo Fisher
    Average 99 stars, based on 75628 article reviews
    Price from $9.99 to $1999.99
    lipofectamine 2000 - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "Amelioration of paraquat-induced pulmonary fibrosis in mice by regulating miR-140-5p expression with the fibrogenic inhibitor Xuebijing"

    Article Title: Amelioration of paraquat-induced pulmonary fibrosis in mice by regulating miR-140-5p expression with the fibrogenic inhibitor Xuebijing

    Journal: International Journal of Immunopathology and Pharmacology

    doi: 10.1177/2058738420923911

    Transfection efficiency and stability of the mimics or inhibitor and effects of miR-140-5p in A549 cells on the Smad/TGF-β1 and Wnt1/β-catenin signaling pathways. miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC were individually mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and placed into the cell culture to transfect A549 cells. After 48 h of transfection, (a) fluorescence expression of transfected cells after miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC transfection (×50); (b) transfection efficiency of miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC; (c) and (d) relative miR-140-5p expression levels were determined by qRT-PCR; (e)–(h) Wnt1, β-catenin, p-Smad3, and TGF-β1 expression levels were measured by Western blot analysis. Data from three independent experiments are presented as mean ± SD. * P
    Figure Legend Snippet: Transfection efficiency and stability of the mimics or inhibitor and effects of miR-140-5p in A549 cells on the Smad/TGF-β1 and Wnt1/β-catenin signaling pathways. miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC were individually mixed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) and placed into the cell culture to transfect A549 cells. After 48 h of transfection, (a) fluorescence expression of transfected cells after miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC transfection (×50); (b) transfection efficiency of miR-140-5p mimics, miR-140-5p mimic-NC, miR-140-5p inhibitor, and miR-140-5p inhibitor-NC; (c) and (d) relative miR-140-5p expression levels were determined by qRT-PCR; (e)–(h) Wnt1, β-catenin, p-Smad3, and TGF-β1 expression levels were measured by Western blot analysis. Data from three independent experiments are presented as mean ± SD. * P

    Techniques Used: Transfection, Cell Culture, Fluorescence, Expressing, Quantitative RT-PCR, Western Blot

    2) Product Images from "Exon-Skipping Oligonucleotides Restore Functional Collagen VI by Correcting a Common COL6A1 Mutation in Ullrich CMD"

    Article Title: Exon-Skipping Oligonucleotides Restore Functional Collagen VI by Correcting a Common COL6A1 Mutation in Ullrich CMD

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2020.05.029

    The Efficiency of ASOs in 2′-MOE Backbone in Pseudo-Exon Skipping at RNA Level (A) Representative picture of PCR products from fibroblasts treated with ASO-5, ASO-6, ASO-5/6, or ASO-9 in 2′-MOE backbone for 24 h with Lipofectamine transfection. (B) Quantitative real-time PCRs of total COL6A1 and mutant COL6A1 transcripts were performed in RNA samples collected from four patient skin fibroblasts treated with ASO-5, ASO-6, ASO-5/6, or ASO-9 at concentrations ranging from 2.5 to 20 nM. Data are presented as mean ± SD. Data were analyzed by one-way ANOVA and post-Bonferroni test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.0001).
    Figure Legend Snippet: The Efficiency of ASOs in 2′-MOE Backbone in Pseudo-Exon Skipping at RNA Level (A) Representative picture of PCR products from fibroblasts treated with ASO-5, ASO-6, ASO-5/6, or ASO-9 in 2′-MOE backbone for 24 h with Lipofectamine transfection. (B) Quantitative real-time PCRs of total COL6A1 and mutant COL6A1 transcripts were performed in RNA samples collected from four patient skin fibroblasts treated with ASO-5, ASO-6, ASO-5/6, or ASO-9 at concentrations ranging from 2.5 to 20 nM. Data are presented as mean ± SD. Data were analyzed by one-way ANOVA and post-Bonferroni test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗∗p ≤ 0.0001).

    Techniques Used: Polymerase Chain Reaction, Allele-specific Oligonucleotide, Transfection, Mutagenesis

    Evaluation of ASOs Specificity and Efficiency in Pseudo-Exon Skipping at the RNA Level (A) Gel electrophoresis of PCR products amplified in RNA samples isolated from UCMD fibroblasts treated with ASOs at 20 nM for 24 h with Lipofectamine transfection. (B) Schematic representation of quantitative real-time PCR assay. Two sets of primers with specific probes were used in the analysis. One set of primers with a specific probe complementary to the pseudo-exon (PE) to exclusively amplify the mutant transcripts. Another set of primers to amplify the total COL6A1 transcripts, including both wild-type and mutant transcripts. (C) Quantitative real-time PCR was performed in RNA samples collected from UCMD fibroblasts treated with 20 nM ASOs after 24 h of transfection, using specific primers and probes. The two regions, where ASOs are capable of efficiently skipping the PE from the mutant transcripts, are underlined by a dashed line in red. Data were normalized to untreated samples and analyzed by one-way ANOVA and post-Bonferroni test. Data are presented as mean ± SD (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).
    Figure Legend Snippet: Evaluation of ASOs Specificity and Efficiency in Pseudo-Exon Skipping at the RNA Level (A) Gel electrophoresis of PCR products amplified in RNA samples isolated from UCMD fibroblasts treated with ASOs at 20 nM for 24 h with Lipofectamine transfection. (B) Schematic representation of quantitative real-time PCR assay. Two sets of primers with specific probes were used in the analysis. One set of primers with a specific probe complementary to the pseudo-exon (PE) to exclusively amplify the mutant transcripts. Another set of primers to amplify the total COL6A1 transcripts, including both wild-type and mutant transcripts. (C) Quantitative real-time PCR was performed in RNA samples collected from UCMD fibroblasts treated with 20 nM ASOs after 24 h of transfection, using specific primers and probes. The two regions, where ASOs are capable of efficiently skipping the PE from the mutant transcripts, are underlined by a dashed line in red. Data were normalized to untreated samples and analyzed by one-way ANOVA and post-Bonferroni test. Data are presented as mean ± SD (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Isolation, Transfection, Real-time Polymerase Chain Reaction, Mutagenesis

    The Efficiency of ASOs on 2′-OMe Backbone in Restoring ECM Collagen VI Protein (A) Patient skin fibroblasts were treated with ASOs at 20 nM using Lipofectamine as transfection reagent. After 24 h, the transfection medium was replaced with growth medium containing l -ascorbic acid (50 μg/mL) for 48 h before being processed for collagen VI immunostaining. (B) Collagen VI protein in the ECM (in green) and nuclei (in blue) were displayed by immunofluorescence staining. Pictures were captured under fluorescence microscopy at 10× (upper panel) and 40× (lower panel) original magnification. Scale bars: 100 μm (upper panel) and 50 μm (lower panel), respectively. (C) Mean intensity and the area covered by collagen VI were quantified in fibroblasts treated with a single transfection of ASO-scr, ASO-3, ASO-4, ASO-5, and ASO-6 at 20 nM for 24 h followed by l -ascorbic acid (50 μg/mL) treatment for 48 h. Data represent mean ± SD from analysis of six individual field images acquired at 40× original magnification under fluorescence microscopy. Data were analyzed by one-way ANOVA and post-Bonferroni test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).
    Figure Legend Snippet: The Efficiency of ASOs on 2′-OMe Backbone in Restoring ECM Collagen VI Protein (A) Patient skin fibroblasts were treated with ASOs at 20 nM using Lipofectamine as transfection reagent. After 24 h, the transfection medium was replaced with growth medium containing l -ascorbic acid (50 μg/mL) for 48 h before being processed for collagen VI immunostaining. (B) Collagen VI protein in the ECM (in green) and nuclei (in blue) were displayed by immunofluorescence staining. Pictures were captured under fluorescence microscopy at 10× (upper panel) and 40× (lower panel) original magnification. Scale bars: 100 μm (upper panel) and 50 μm (lower panel), respectively. (C) Mean intensity and the area covered by collagen VI were quantified in fibroblasts treated with a single transfection of ASO-scr, ASO-3, ASO-4, ASO-5, and ASO-6 at 20 nM for 24 h followed by l -ascorbic acid (50 μg/mL) treatment for 48 h. Data represent mean ± SD from analysis of six individual field images acquired at 40× original magnification under fluorescence microscopy. Data were analyzed by one-way ANOVA and post-Bonferroni test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).

    Techniques Used: Transfection, Immunostaining, Immunofluorescence, Staining, Fluorescence, Microscopy, Allele-specific Oligonucleotide

    3) Product Images from "Polymorphic α-Synuclein Strains Modified by Dopamine and Docosahexaenoic Acid Interact Differentially with Tau Protein"

    Article Title: Polymorphic α-Synuclein Strains Modified by Dopamine and Docosahexaenoic Acid Interact Differentially with Tau Protein

    Journal: Molecular Neurobiology

    doi: 10.1007/s12035-020-01913-6

    Seeding assay of tau aggregate strains in Tau RD-P301S CFP/YFP FRET biosensor cells. a – d Tau biosensor cells were exposed to the cross-seeded tau aggregates or unseeded tau aggregate at 0.25 and 0.5 μM concentrations for 24 h with Lipofectamine. The cross-seeded tau aggregates, TauO SynO-DA and TauO SynO-DHA show dose-dependent increased seeding propensity at the concentrations used resulting in the formation of cytosolic tau aggregates. Unseeded tau aggregates do not show any seeding. TauO SynO-DA has more potent seeding efficiency than TauO SynO-DHA . e Quantification of FRET positive cells observed in all 4 groups of treatment: vehicle, TauO unseeded , TauO SynO-DA , and TauO SynO-DHA . Quantification is performed from fifteen regions of interest (ROIs) from three replicates performed in three independent experiments. The histograms represent mean ± SD. Statistical significance is measured by using two-way ANOVA with Bonferroni post hoc analysis. ** p
    Figure Legend Snippet: Seeding assay of tau aggregate strains in Tau RD-P301S CFP/YFP FRET biosensor cells. a – d Tau biosensor cells were exposed to the cross-seeded tau aggregates or unseeded tau aggregate at 0.25 and 0.5 μM concentrations for 24 h with Lipofectamine. The cross-seeded tau aggregates, TauO SynO-DA and TauO SynO-DHA show dose-dependent increased seeding propensity at the concentrations used resulting in the formation of cytosolic tau aggregates. Unseeded tau aggregates do not show any seeding. TauO SynO-DA has more potent seeding efficiency than TauO SynO-DHA . e Quantification of FRET positive cells observed in all 4 groups of treatment: vehicle, TauO unseeded , TauO SynO-DA , and TauO SynO-DHA . Quantification is performed from fifteen regions of interest (ROIs) from three replicates performed in three independent experiments. The histograms represent mean ± SD. Statistical significance is measured by using two-way ANOVA with Bonferroni post hoc analysis. ** p

    Techniques Used:

    4) Product Images from "Effects of emodin on intestinal mucosal barrier by the upregulation of miR-218a-5p expression in rats with acute necrotizing pancreatitis"

    Article Title: Effects of emodin on intestinal mucosal barrier by the upregulation of miR-218a-5p expression in rats with acute necrotizing pancreatitis

    Journal: International Journal of Immunopathology and Pharmacology

    doi: 10.1177/2058738420941765

    Effect of miR-218-5p on the expression of Bax, Fas, Bcl-2, caspase-3, Rock1, and Notch1 and cell apoptosis in rat intestinal epithelial cells. miR-218-5p mimics, miR-218-5p mimic-negative control (NC), miR-218-5p inhibitor, and miR-218-5p inhibitor-NC were mixed with Lipofectamine 2000 (Invitrogen) and then added to the cell culture. After 24 h of transfection, (a, b) the expression levels of Bax, caspase-3, Fas, Rock1, and Notch1 were measured by Western blot; (c, d) the level of miR-218-5p was measured by RT-PCR; and (e–i) the apoptosis of rat intestinal epithelial cells was measured by flow cytometry. (e) Typical flowchart of rat intestinal epithelial cell apoptosis and (i) the apoptosis rate of rat intestinal epithelial cells by flow cytometry analysis. Data obtained through quantitative densitometry are presented as mean ± SD of three independent experiments. * P
    Figure Legend Snippet: Effect of miR-218-5p on the expression of Bax, Fas, Bcl-2, caspase-3, Rock1, and Notch1 and cell apoptosis in rat intestinal epithelial cells. miR-218-5p mimics, miR-218-5p mimic-negative control (NC), miR-218-5p inhibitor, and miR-218-5p inhibitor-NC were mixed with Lipofectamine 2000 (Invitrogen) and then added to the cell culture. After 24 h of transfection, (a, b) the expression levels of Bax, caspase-3, Fas, Rock1, and Notch1 were measured by Western blot; (c, d) the level of miR-218-5p was measured by RT-PCR; and (e–i) the apoptosis of rat intestinal epithelial cells was measured by flow cytometry. (e) Typical flowchart of rat intestinal epithelial cell apoptosis and (i) the apoptosis rate of rat intestinal epithelial cells by flow cytometry analysis. Data obtained through quantitative densitometry are presented as mean ± SD of three independent experiments. * P

    Techniques Used: Expressing, Negative Control, Cell Culture, Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry

    5) Product Images from "Nurr1 performs its anti-inflammatory function by regulating RasGRP1 expression in neuro-inflammation"

    Article Title: Nurr1 performs its anti-inflammatory function by regulating RasGRP1 expression in neuro-inflammation

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67549-7

    Nurr1 repressed the transcriptional activity of the  RasGRP1  gene. ( A ) Schematic representation of the mouse  RasGRP1  gene, indicating the position of NBRE in the  RasGRP1  intron. The second intron region in the  RasGRP1  gene contains the NBRE (AAAGGTCA) sequence. The  black boxes  represent the exons. ( B ) Schematic diagram of the luciferase reporter construct used in the assays. ( C ) 293 T cells were transfected with either the reporter plasmid alone (250 ng) or a combination of different amounts of the Nurr1 expression vector (50, 100, or 250 ng) using Lipofectamine 2000. After 24 h of transfection, the cells were harvested and lysed, and the luciferase activity was quantified with the dual-luciferase assay system.  Renilla  activity was used for normalization. This experiment was repeated three times using independently prepared cell lysates. Statistical analysis was performed using Student's t-test (mean ± SD; n = 3; **P 
    Figure Legend Snippet: Nurr1 repressed the transcriptional activity of the RasGRP1 gene. ( A ) Schematic representation of the mouse RasGRP1 gene, indicating the position of NBRE in the RasGRP1 intron. The second intron region in the RasGRP1 gene contains the NBRE (AAAGGTCA) sequence. The black boxes represent the exons. ( B ) Schematic diagram of the luciferase reporter construct used in the assays. ( C ) 293 T cells were transfected with either the reporter plasmid alone (250 ng) or a combination of different amounts of the Nurr1 expression vector (50, 100, or 250 ng) using Lipofectamine 2000. After 24 h of transfection, the cells were harvested and lysed, and the luciferase activity was quantified with the dual-luciferase assay system. Renilla activity was used for normalization. This experiment was repeated three times using independently prepared cell lysates. Statistical analysis was performed using Student's t-test (mean ± SD; n = 3; **P 

    Techniques Used: Activity Assay, Sequencing, Luciferase, Construct, Transfection, Plasmid Preparation, Expressing

    6) Product Images from "Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists"

    Article Title: Rational design of antisense oligonucleotides modulating the activity of TLR7/8 agonists

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa523

    Rational selection of HPRT -targeting ASOs exhibiting TLR8 potentiation. ( A ) HeLa cells were reverse-transfected with indicated HPRT -targeting ASO quantities as detailed in the Materials and Methods section and Supplementary Table S3 , and HPRT levels measured by RT-qPCR after 24h incubation. HPRT levels were reported to SFRS9 expression, and further normalised to the average of the non-targeting ASONC1 and ASONC5 control conditions. Data shown represent the average of three independent experiments conducted in biological triplicate (± s.e.m). (B, C) HEK-TLR7 ( B ) and HEK-TLR8 ( C ) cells expressing an NF-κB-luciferase reporter were treated with 500 nM ASOs for 20 min prior to stimulation with 1 μg/ml R848. NF-κB-luciferase levels were measured after overnight incubation. Percentages (B) or fold increases (C) relative to the condition ‘R848 without ASO’ (B) or NT condition (C) are averaged from three independent experiments in biological triplicate (± s.e.m and ordinary one-way ANOVA with Dunnett's multiple comparison tests to the NT condition [B] or the ‘R848 without ASO’ condition [C] are shown). ( D ) Selected HPRT ASO sequences with low TLR7 inhibition. The conserved region between all the sequences is highlighted in green. The 2′OMe flanking regions are highlighted in orange. CUU motifs are in bold blue, while UUC motifs are underlined. (E, F) WT THP-1 were incubated overnight with 100 nM ASOs. The next day, the cells were treated with lipofectamine 2000 (at 2.5 μl/ml, to enhance cytoplasmic delivery of the ASOs), just before R848 stimulation (1 μg/ml for F only). Supernatants were collected after 8 h for IP-10 ELISA ( F ), and cells lysed for RNA purification after 24 h ( E ). (E) HPRT levels were reported to 18S , and normalised to NT condition. Data are averaged from four (E) or three (F) independent experiments in duplicate (± s.e.m. and ordinary one-way ANOVA with Dunnett's multiple comparison tests to the ‘R848 without ASO’ condition [F] or Mann–Whitney U tests [E] are shown). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns: non-significant.
    Figure Legend Snippet: Rational selection of HPRT -targeting ASOs exhibiting TLR8 potentiation. ( A ) HeLa cells were reverse-transfected with indicated HPRT -targeting ASO quantities as detailed in the Materials and Methods section and Supplementary Table S3 , and HPRT levels measured by RT-qPCR after 24h incubation. HPRT levels were reported to SFRS9 expression, and further normalised to the average of the non-targeting ASONC1 and ASONC5 control conditions. Data shown represent the average of three independent experiments conducted in biological triplicate (± s.e.m). (B, C) HEK-TLR7 ( B ) and HEK-TLR8 ( C ) cells expressing an NF-κB-luciferase reporter were treated with 500 nM ASOs for 20 min prior to stimulation with 1 μg/ml R848. NF-κB-luciferase levels were measured after overnight incubation. Percentages (B) or fold increases (C) relative to the condition ‘R848 without ASO’ (B) or NT condition (C) are averaged from three independent experiments in biological triplicate (± s.e.m and ordinary one-way ANOVA with Dunnett's multiple comparison tests to the NT condition [B] or the ‘R848 without ASO’ condition [C] are shown). ( D ) Selected HPRT ASO sequences with low TLR7 inhibition. The conserved region between all the sequences is highlighted in green. The 2′OMe flanking regions are highlighted in orange. CUU motifs are in bold blue, while UUC motifs are underlined. (E, F) WT THP-1 were incubated overnight with 100 nM ASOs. The next day, the cells were treated with lipofectamine 2000 (at 2.5 μl/ml, to enhance cytoplasmic delivery of the ASOs), just before R848 stimulation (1 μg/ml for F only). Supernatants were collected after 8 h for IP-10 ELISA ( F ), and cells lysed for RNA purification after 24 h ( E ). (E) HPRT levels were reported to 18S , and normalised to NT condition. Data are averaged from four (E) or three (F) independent experiments in duplicate (± s.e.m. and ordinary one-way ANOVA with Dunnett's multiple comparison tests to the ‘R848 without ASO’ condition [F] or Mann–Whitney U tests [E] are shown). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ns: non-significant.

    Techniques Used: Selection, Transfection, Allele-specific Oligonucleotide, Quantitative RT-PCR, Incubation, Expressing, Luciferase, Inhibition, Enzyme-linked Immunosorbent Assay, Purification, MANN-WHITNEY

    7) Product Images from "Aβ and tau prion-like activities decline with longevity in Alzheimer’s disease brains"

    Article Title: Aβ and tau prion-like activities decline with longevity in Alzheimer’s disease brains

    Journal: Science translational medicine

    doi: 10.1126/scitranslmed.aat8462

    Development of YFP-Aβ fusion proteins in HEK-293T cell lines for measuring Aβ aggregates in brains. The indicated cell lines were developed to measure prion-like activity of preparations consisting of synthetic Aβ peptides and mouse brain-derived extracts based on their abilities to induce fluorescent aggregates which we refer to as puncta. ( A ) Diagram illustrating the Aβ constructs used in this study (left). Stably transfected HEK293T cells expressing an Aβ-YFP fusion construct underwent lipofectamine-based transduction with synthetic Aβ fibrils (right). ( B ) Representative confocal images of HEK293T cells expressing Aβ42 fused to YFP at the N-terminus (clone #1), which were treated with PBS (left; control) or exposed to synthetic Aβ40 fibrils (initial monomeric concentration, 1 μM) (right, exposed). The aggregates of Aβ-YFP appear as fluorescent yellow puncta. To measure prion-like activity, we counted the number of puncta-positive cells and expressed this as a percent of the total number of cells in the field of view (% positive cells). Lower panels are higher magnification images of white boxed areas in upper panels. Scale bars: 20 μm (upper panels) or 5 μm (lower panels). ( C ) HEK293T cells transfected with YFP-Aβ42 were treated with two different types of Aβ ranging from 0.03–100 nM (initial monomeric concentration): synthetic Aβ40 (left) or Aβ purified from TgAPP23 mouse brains (right). Puncta-inducing activity in the HEK293T cells was quantified 2 days after the initial exposure to various Aβ preparations. Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 3 independent experiments. ( D ) Cell lines stably expressing four different wildtype (WT) Aβ constructs (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, , and are representative of 2 to 3 independent experiments. ( E ) Cell lines stably expressing four different Aβ40 constructs fused to YFP at the N-terminus (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 2 independent experiments.
    Figure Legend Snippet: Development of YFP-Aβ fusion proteins in HEK-293T cell lines for measuring Aβ aggregates in brains. The indicated cell lines were developed to measure prion-like activity of preparations consisting of synthetic Aβ peptides and mouse brain-derived extracts based on their abilities to induce fluorescent aggregates which we refer to as puncta. ( A ) Diagram illustrating the Aβ constructs used in this study (left). Stably transfected HEK293T cells expressing an Aβ-YFP fusion construct underwent lipofectamine-based transduction with synthetic Aβ fibrils (right). ( B ) Representative confocal images of HEK293T cells expressing Aβ42 fused to YFP at the N-terminus (clone #1), which were treated with PBS (left; control) or exposed to synthetic Aβ40 fibrils (initial monomeric concentration, 1 μM) (right, exposed). The aggregates of Aβ-YFP appear as fluorescent yellow puncta. To measure prion-like activity, we counted the number of puncta-positive cells and expressed this as a percent of the total number of cells in the field of view (% positive cells). Lower panels are higher magnification images of white boxed areas in upper panels. Scale bars: 20 μm (upper panels) or 5 μm (lower panels). ( C ) HEK293T cells transfected with YFP-Aβ42 were treated with two different types of Aβ ranging from 0.03–100 nM (initial monomeric concentration): synthetic Aβ40 (left) or Aβ purified from TgAPP23 mouse brains (right). Puncta-inducing activity in the HEK293T cells was quantified 2 days after the initial exposure to various Aβ preparations. Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 3 independent experiments. ( D ) Cell lines stably expressing four different wildtype (WT) Aβ constructs (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, , and are representative of 2 to 3 independent experiments. ( E ) Cell lines stably expressing four different Aβ40 constructs fused to YFP at the N-terminus (shown in panel A ). Data shown are mean ± SEM as determined from four images per well across four wells, and are representative of 2 independent experiments.

    Techniques Used: Activity Assay, Derivative Assay, Construct, Stable Transfection, Transfection, Expressing, Transduction, Concentration Assay, Purification

    8) Product Images from "Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines"

    Article Title: Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134396

    ERβ, but not ERα, is expressed in human melanoma cells. (A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.
    Figure Legend Snippet: ERβ, but not ERα, is expressed in human melanoma cells. (A) As a positive control, the expression of ERβ was evaluated by Western blot analysis in human BLM melanoma cells engineered to overexpress the receptor subtype protein, utilizing two primary antibodies: H-150 (Santa Cruz) and 14C8 (Abcam). A band corresponding to the receptor protein (59 kDa) was detected in basal conditions, both in control (C) and in Lipofectamine (Lipo) treated BLM cells. As expected, the intensity of this band was found to be significantly increased after ERβ overexpression (24–72 h), with the highest level of expression at 24 h. (B) By Western blot analysis, utilizing the two primary antibodies H-150 and 14C8, ERβ was found to be expressed at high levels in human BLM, A375, WM115, WM1552 melanoma cell lines (lanes 3, 4, 5, 7), while the human IGR-39 melanoma cell line expressed the receptor at almost undetectable levels (lane 6). ERβ was also expressed in human MCF-7 breast cancer cells, utilized as a positive control (lane 1), but it was not expressed in the human HEK293, utilized as a negative control. (C) On the other hand, all the human melanoma cells lines tested (lanes 2–6) did not express the ERα receptor isoform, which was expressed only in the control cell line (MCF-7, lane 1). β-actin was utilized as a loading control. For each analysis, one representative of three different experiments, which gave similar results, is shown.

    Techniques Used: Positive Control, Expressing, Western Blot, Over Expression, Negative Control

    9) Product Images from "Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate"

    Article Title: Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2476-2

    Either BAK or BAX knockdown decreases TNFα+CHX or navitoclax-induced pyroptosis. a Efficiency of BAK and/or BAX knockdown was detected by immunoblotting. b–e After BAK siRNA, BAX siRNA, BAK siRNA+BAX siRNA, or negative control siRNA were transfected into HCT116 cells by Lipofectamine RNAiMAX, cells were reseeded into 24-well plate followed by treatment of TNFα+CHX ( b , d ) or navitoclax ( c , e ) for 24 h. Culture supernatants were collected to measure the percentage of LDH release ( b , c ) and phase contrast images were taken ( d , e ) at the indicated time points.
    Figure Legend Snippet: Either BAK or BAX knockdown decreases TNFα+CHX or navitoclax-induced pyroptosis. a Efficiency of BAK and/or BAX knockdown was detected by immunoblotting. b–e After BAK siRNA, BAX siRNA, BAK siRNA+BAX siRNA, or negative control siRNA were transfected into HCT116 cells by Lipofectamine RNAiMAX, cells were reseeded into 24-well plate followed by treatment of TNFα+CHX ( b , d ) or navitoclax ( c , e ) for 24 h. Culture supernatants were collected to measure the percentage of LDH release ( b , c ) and phase contrast images were taken ( d , e ) at the indicated time points.

    Techniques Used: Negative Control, Transfection

    10) Product Images from "Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy"

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy

    Journal: Molecules

    doi: 10.3390/molecules25081994

    Colony survival assay in A549 cells following transfection with Lipofectamine 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.
    Figure Legend Snippet: Colony survival assay in A549 cells following transfection with Lipofectamine 2000 ( A ) or PEI-PCL-PEG (PPP) micelleplexes ( B ). Untreated (open circle), negative control (filled triangles) or ERCC1-XPF (filled squares) siRNA transfected cells were treated with increasing doses of cisplatin for 2 h, and cell viability was determined by a clonogenic assay. Results are represented as mean ± SD. IC 50 values were calculated using Compusyn software.

    Techniques Used: Clonogenic Cell Survival Assay, Transfection, Negative Control, Clonogenic Assay, Software

    ERCC1 and XPF gene knockdown efficiency was validated in lung adenocarcinoma cells (A549) after 72 h following double transfection of Lipofectamine 2000 (LF) or micelleplexes (PPP) with 100 pmol ERCC1-XPF siRNA or negative control (NC) siRNA. ERCC1 and XPF expression was normalized with GAPDH expression and quantified by real time PCR. Data points indicate mean ± SD. (n = 6).
    Figure Legend Snippet: ERCC1 and XPF gene knockdown efficiency was validated in lung adenocarcinoma cells (A549) after 72 h following double transfection of Lipofectamine 2000 (LF) or micelleplexes (PPP) with 100 pmol ERCC1-XPF siRNA or negative control (NC) siRNA. ERCC1 and XPF expression was normalized with GAPDH expression and quantified by real time PCR. Data points indicate mean ± SD. (n = 6).

    Techniques Used: Transfection, Negative Control, Expressing, Real-time Polymerase Chain Reaction

    Western Blot analysis of excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum group F (XPF) protein levels within A549 cells following ( A ) single transfection of micelleplexes (PPP) loaded with 50 pmol small interfering RNA (siRNA) after 72 h and ( B ) double transfection of micelleplexes (PPP) loaded with 100 pmol siRNA after 72h. Lipofectamine 2000 (LF) lipoplexes were prepared according to manufacturer’s protocol with 50 pmol (A) and 100 pmol (B) siRNA (n = 2).
    Figure Legend Snippet: Western Blot analysis of excision repair cross-complementation group 1 (ERCC1) and xeroderma pigmentosum group F (XPF) protein levels within A549 cells following ( A ) single transfection of micelleplexes (PPP) loaded with 50 pmol small interfering RNA (siRNA) after 72 h and ( B ) double transfection of micelleplexes (PPP) loaded with 100 pmol siRNA after 72h. Lipofectamine 2000 (LF) lipoplexes were prepared according to manufacturer’s protocol with 50 pmol (A) and 100 pmol (B) siRNA (n = 2).

    Techniques Used: Western Blot, Transfection, Small Interfering RNA

    11) Product Images from "Mitochondrial-targeted Aryl Hydrocarbon Receptor and the Impact of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Cellular Respiration and the Mitochondrial Proteome"

    Article Title: Mitochondrial-targeted Aryl Hydrocarbon Receptor and the Impact of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Cellular Respiration and the Mitochondrial Proteome

    Journal: Toxicology and applied pharmacology

    doi: 10.1016/j.taap.2016.04.005

    The effect of AIP knockdown on the amount of AHR in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown AIP in Hepa1c1c7 cells. Western blot analysis for AIP and AHR expression was performed from whole cell lysate, and nuclear, cytosolic, and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction, and cytochrome c oxidase subunit IV (COX4) was a loading control for the mitochondrial fraction. (A) Results shown are representative of 3 independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM Silencer® Select Negative Control #1 siRNA (siCtrl), lane 3: 10 nM siRNA1 for AIP (siAIP1), lane4: 10 nM siRNA2 for AIP (siAIP2). (B) Densitometry was determined with a Fuji Image analyzer. The levels of expressions of AIP and AHR protein were normalized to each loading control. The normalized protein expression levels were compared with each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).
    Figure Legend Snippet: The effect of AIP knockdown on the amount of AHR in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown AIP in Hepa1c1c7 cells. Western blot analysis for AIP and AHR expression was performed from whole cell lysate, and nuclear, cytosolic, and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction, and cytochrome c oxidase subunit IV (COX4) was a loading control for the mitochondrial fraction. (A) Results shown are representative of 3 independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM Silencer® Select Negative Control #1 siRNA (siCtrl), lane 3: 10 nM siRNA1 for AIP (siAIP1), lane4: 10 nM siRNA2 for AIP (siAIP2). (B) Densitometry was determined with a Fuji Image analyzer. The levels of expressions of AIP and AHR protein were normalized to each loading control. The normalized protein expression levels were compared with each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).

    Techniques Used: Western Blot, Expressing, Negative Control

    The effect of TOMM20 knockdown on the amount of AHR protein in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown TOMM20 in Hepa1c1c7 cells. Western blot analysis for TOMM20 and AHR expression was performed from whole cell lysate, and nuclear, cytosolic and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction and ATP5α was a loading control for the mitochondrial fraction. (A) Results shown are representative of three independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM nontargeting siRNA (siCtrl), lane 3: 10 nM siRNA for TOMM20 (siTomm20). (B and C) Densitometry was determined with a Fuji Image analyzer. The expressions of TOMM20 and AHR protein were normalized to each loading control. The normalized protein expression levels were compared to each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).
    Figure Legend Snippet: The effect of TOMM20 knockdown on the amount of AHR protein in cellular fractions of hepa1c1c7 cells siRNA was used to knockdown TOMM20 in Hepa1c1c7 cells. Western blot analysis for TOMM20 and AHR expression was performed from whole cell lysate, and nuclear, cytosolic and mitochondrial fractions. α-tubulin was used as a loading control for the whole cell lysate, histone H3 (H3) was a loading control for the nuclear fraction, lactate dehydrogenase (LDH) was a loading control for the cytosolic fraction and ATP5α was a loading control for the mitochondrial fraction. (A) Results shown are representative of three independent experiments. Lane 1: lipofectamine treatment (Lipomock), lane 2: 10 nM nontargeting siRNA (siCtrl), lane 3: 10 nM siRNA for TOMM20 (siTomm20). (B and C) Densitometry was determined with a Fuji Image analyzer. The expressions of TOMM20 and AHR protein were normalized to each loading control. The normalized protein expression levels were compared to each other by re-normalization with the protein expression from which cells were affected by lipofectamine only (Lipomock). The bars represent mean ± the standard errors (n=3).

    Techniques Used: Western Blot, Expressing

    12) Product Images from "Microvesicles from malaria-infected red blood cells activate natural killer cells via MDA5 pathway"

    Article Title: Microvesicles from malaria-infected red blood cells activate natural killer cells via MDA5 pathway

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1007298

    Requirement of MDA5 in NK cell responses to iRBCs. (A) Heatmap of DEGs involved in RLR signaling. Each column represents an individual of the indicated group. (B-C) NK cells were co-cultured with iRBCs for 96 h, and intracellular staining of MDA5 was analyzed by flow cytometry. Shown are representative histogram (B) and MFI (C) of MDA5 staining of R-NK and NR-NK cells. (D) Western blot of MDA5 and GAPDH levels in R-NK cells transduced with lentivirus expressing CRISPR/Cas9 and a gRNA targeting either MDA5 (gMDA5) or a scramble sequence (gScrble). (E-F) Transduced NK cells were co-cultured with iRBCs for 48 h and parasitemia was quantified. Representative Hoechst vs CD56 staining profiles of iRBCs alone, iRBCs co-cultured with R-NK cells with or without MDA5 knockdown (E) and comparison of reduction in parasitemia (F) . Numbers in E indicate parasitemia. (G-H) R-NK cells were co-cultured with either lipofectamine or varying concentrations of lipofectamine-formulated (Lf)-poly I:C for 48 h. Surface expression of CD69 was determined by flow cytometry (G) , and IFN-γ secretion was determined by immunoplex assay (H) . (I) iRBCs were cultured alone or with either R-NK or NR-NK cells in the presence of 1000μg/ml lipofectamine-formulated poly I:C for 96 h. Parasitemia was quantified by flow cytometry. Representative Hoechst vs CD56 staining profiles is shown. (J) R-NK and NR-NK cells were co-cultured with iRBCs under the indicated conditions for 96 h, and reduction in parasitemia was quantified by flow cytometry. Poly I:C was used at 100 μg/ml (poly I:C-100). poly I:C (Lf) was used at 100, 10 and 1 μg/ml. (K) NR-NK were co-cultured with iRBCs with or without 5’pppRNA (Lf) (10 μg/ml), and reduction in parasitemia was quantified by flow cytometry. Each symbol represents a different individual. Error bars represent mean ± SD. *p
    Figure Legend Snippet: Requirement of MDA5 in NK cell responses to iRBCs. (A) Heatmap of DEGs involved in RLR signaling. Each column represents an individual of the indicated group. (B-C) NK cells were co-cultured with iRBCs for 96 h, and intracellular staining of MDA5 was analyzed by flow cytometry. Shown are representative histogram (B) and MFI (C) of MDA5 staining of R-NK and NR-NK cells. (D) Western blot of MDA5 and GAPDH levels in R-NK cells transduced with lentivirus expressing CRISPR/Cas9 and a gRNA targeting either MDA5 (gMDA5) or a scramble sequence (gScrble). (E-F) Transduced NK cells were co-cultured with iRBCs for 48 h and parasitemia was quantified. Representative Hoechst vs CD56 staining profiles of iRBCs alone, iRBCs co-cultured with R-NK cells with or without MDA5 knockdown (E) and comparison of reduction in parasitemia (F) . Numbers in E indicate parasitemia. (G-H) R-NK cells were co-cultured with either lipofectamine or varying concentrations of lipofectamine-formulated (Lf)-poly I:C for 48 h. Surface expression of CD69 was determined by flow cytometry (G) , and IFN-γ secretion was determined by immunoplex assay (H) . (I) iRBCs were cultured alone or with either R-NK or NR-NK cells in the presence of 1000μg/ml lipofectamine-formulated poly I:C for 96 h. Parasitemia was quantified by flow cytometry. Representative Hoechst vs CD56 staining profiles is shown. (J) R-NK and NR-NK cells were co-cultured with iRBCs under the indicated conditions for 96 h, and reduction in parasitemia was quantified by flow cytometry. Poly I:C was used at 100 μg/ml (poly I:C-100). poly I:C (Lf) was used at 100, 10 and 1 μg/ml. (K) NR-NK were co-cultured with iRBCs with or without 5’pppRNA (Lf) (10 μg/ml), and reduction in parasitemia was quantified by flow cytometry. Each symbol represents a different individual. Error bars represent mean ± SD. *p

    Techniques Used: Cell Culture, Staining, Flow Cytometry, Cytometry, Western Blot, Transduction, Expressing, CRISPR, Sequencing

    13) Product Images from "OFD1, as a Ciliary Protein, Exhibits Neuroprotective Function in Photoreceptor Degeneration Models"

    Article Title: OFD1, as a Ciliary Protein, Exhibits Neuroprotective Function in Photoreceptor Degeneration Models

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155860

    Ofd1 protection of photoreceptors from oxidative stress via decreasing ROS production. (A) The intracellular oxidant DCFH of MNU-induced ROS in 661W cells observed by green fluorescence using Leica fluorescence microscope (with monochromatic CCD), and measured using fluorometer at wavelength of 488/525 nm. ROS production was stimulated 10 mins by 500 ug/ml MNU or ROSup as positive control. N: non-treated cells. MNU: MNU 500 ug/ml treated 661W for 10 mins. MNU+Empty Vector: after 661W transfected with pEGFP vector for 48h, MNU 500 ug/ml treated for 10 mins. MNU+Ofd1 CDS: after 661W transfected with pEGFP-Ofd1-CDS for 48h, MNU 500 ug/ml treated for 10 mins. Scale bar: 50μm. (B) Cell viability and proliferation detection in 661W cell using the MTT assay. Ofd1 expression was up- or down-regulated by plasmid transfection by Lipofectamine 2000. Ofd1-CDS was used to overexpress Ofd1, and Ofd1-shRNA was used to inhibit expression. After transfection for 36 h, 500 ug/ml MNU was added and incubated for additional 12 h. NC: normal control. * p
    Figure Legend Snippet: Ofd1 protection of photoreceptors from oxidative stress via decreasing ROS production. (A) The intracellular oxidant DCFH of MNU-induced ROS in 661W cells observed by green fluorescence using Leica fluorescence microscope (with monochromatic CCD), and measured using fluorometer at wavelength of 488/525 nm. ROS production was stimulated 10 mins by 500 ug/ml MNU or ROSup as positive control. N: non-treated cells. MNU: MNU 500 ug/ml treated 661W for 10 mins. MNU+Empty Vector: after 661W transfected with pEGFP vector for 48h, MNU 500 ug/ml treated for 10 mins. MNU+Ofd1 CDS: after 661W transfected with pEGFP-Ofd1-CDS for 48h, MNU 500 ug/ml treated for 10 mins. Scale bar: 50μm. (B) Cell viability and proliferation detection in 661W cell using the MTT assay. Ofd1 expression was up- or down-regulated by plasmid transfection by Lipofectamine 2000. Ofd1-CDS was used to overexpress Ofd1, and Ofd1-shRNA was used to inhibit expression. After transfection for 36 h, 500 ug/ml MNU was added and incubated for additional 12 h. NC: normal control. * p

    Techniques Used: Fluorescence, Microscopy, Positive Control, Plasmid Preparation, Transfection, MTT Assay, Expressing, shRNA, Incubation

    14) Product Images from "Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening"

    Article Title: Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197373

    Screening of 3-mix LNA-based SSO cocktails using the established reporter cell line. A) Phase contrast and fluorescence microscopy images of SSO-transfected reporter cells; 21-mix SSOs (100 nM total), 3-mix (02+20+08) SSOs (100 nM total), and mock-transfected cells were analyzed using a BZ-8000 microscope. Fluorescence microscopy, showing that only SSO-transfected reporter cells display red fluorescence. Phase contrast: phase contrast images, TagBFP: blue fluorescence images using the (Ex/Em = 360/460 nm) filter, EGFP: green fluorescence images using the (Ex/Em = 470/525 nm) filter, and TagRFP: red fluorescence images using the (Ex/Em = 545/605 nm) filter. Mock: treated with Lipofectamine 2000 only. Each analysis was duplicated and repeated five times to ensure the reproducibility of the results. B) TagRFP expression levels, measured by HCS. The graph shows the normalized red fluorescence intensity, relative to the value in the 21-mix SSO-transfected cells (set at 1). Values represent the mean ± standard deviation of five independent experiments performed in duplicate. Data were analyzed by ANOVA ( P
    Figure Legend Snippet: Screening of 3-mix LNA-based SSO cocktails using the established reporter cell line. A) Phase contrast and fluorescence microscopy images of SSO-transfected reporter cells; 21-mix SSOs (100 nM total), 3-mix (02+20+08) SSOs (100 nM total), and mock-transfected cells were analyzed using a BZ-8000 microscope. Fluorescence microscopy, showing that only SSO-transfected reporter cells display red fluorescence. Phase contrast: phase contrast images, TagBFP: blue fluorescence images using the (Ex/Em = 360/460 nm) filter, EGFP: green fluorescence images using the (Ex/Em = 470/525 nm) filter, and TagRFP: red fluorescence images using the (Ex/Em = 545/605 nm) filter. Mock: treated with Lipofectamine 2000 only. Each analysis was duplicated and repeated five times to ensure the reproducibility of the results. B) TagRFP expression levels, measured by HCS. The graph shows the normalized red fluorescence intensity, relative to the value in the 21-mix SSO-transfected cells (set at 1). Values represent the mean ± standard deviation of five independent experiments performed in duplicate. Data were analyzed by ANOVA ( P

    Techniques Used: Fluorescence, Microscopy, Transfection, Expressing, Standard Deviation

    15) Product Images from "Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening"

    Article Title: Construction of a tri-chromatic reporter cell line for the rapid and simple screening of splice-switching oligonucleotides targeting DMD exon 51 using high content screening

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0197373

    Screening of 3-mix LNA-based SSO cocktails using the established reporter cell line. A) Phase contrast and fluorescence microscopy images of SSO-transfected reporter cells; 21-mix SSOs (100 nM total), 3-mix (02+20+08) SSOs (100 nM total), and mock-transfected cells were analyzed using a BZ-8000 microscope. Fluorescence microscopy, showing that only SSO-transfected reporter cells display red fluorescence. Phase contrast: phase contrast images, TagBFP: blue fluorescence images using the (Ex/Em = 360/460 nm) filter, EGFP: green fluorescence images using the (Ex/Em = 470/525 nm) filter, and TagRFP: red fluorescence images using the (Ex/Em = 545/605 nm) filter. Mock: treated with Lipofectamine 2000 only. Each analysis was duplicated and repeated five times to ensure the reproducibility of the results. B) TagRFP expression levels, measured by HCS. The graph shows the normalized red fluorescence intensity, relative to the value in the 21-mix SSO-transfected cells (set at 1). Values represent the mean ± standard deviation of five independent experiments performed in duplicate. Data were analyzed by ANOVA ( P
    Figure Legend Snippet: Screening of 3-mix LNA-based SSO cocktails using the established reporter cell line. A) Phase contrast and fluorescence microscopy images of SSO-transfected reporter cells; 21-mix SSOs (100 nM total), 3-mix (02+20+08) SSOs (100 nM total), and mock-transfected cells were analyzed using a BZ-8000 microscope. Fluorescence microscopy, showing that only SSO-transfected reporter cells display red fluorescence. Phase contrast: phase contrast images, TagBFP: blue fluorescence images using the (Ex/Em = 360/460 nm) filter, EGFP: green fluorescence images using the (Ex/Em = 470/525 nm) filter, and TagRFP: red fluorescence images using the (Ex/Em = 545/605 nm) filter. Mock: treated with Lipofectamine 2000 only. Each analysis was duplicated and repeated five times to ensure the reproducibility of the results. B) TagRFP expression levels, measured by HCS. The graph shows the normalized red fluorescence intensity, relative to the value in the 21-mix SSO-transfected cells (set at 1). Values represent the mean ± standard deviation of five independent experiments performed in duplicate. Data were analyzed by ANOVA ( P

    Techniques Used: Fluorescence, Microscopy, Transfection, Expressing, Standard Deviation

    16) Product Images from "Analysis of intermolecular RNA–RNA recombination by rubella virus"

    Article Title: Analysis of intermolecular RNA–RNA recombination by rubella virus

    Journal: Virology

    doi: 10.1016/S0042-6822(03)00064-3

    Recombination in the J-UTR. Genomic diagrams of pairs of replicating and nonreplicating transcripts are shown in (A) that either failed to yield recombinants (NO RECOMBINATION) or yielded recombinants (RECOMBINATION) when Lipofectamine was used as the transfection reagent. Brackets indicate nonreplicating transcripts used individually in cotransfections with a common replicating transcript with a similar result. In some cases, transcript pairs that failed to yield recombinants following Lipofectaine-mediated transfection did yield recombinants when Lipofectamine 2000 was used, as indicated by an asterisk. To determine whether the J-UTR was a recombinational hot spot, DI-Stu and NR-5355-3′ transcripts generated from constructs that had been genetically tagged with restriction sites ( Nsi I at the 5′ end of the J-UTR or Xba I at the 3′ end of the J-UTR) were used in cotransfections (Lipofectamine 2000) and virus recovered from recombinant plaques was sequenced across the region of overlap in the middle of the genome. As shown in (B), recombinants were recovered that had no exchange of the parental markers, exchange of parental markers, and duplications of the 3′ NS-ORF and J-UTR.
    Figure Legend Snippet: Recombination in the J-UTR. Genomic diagrams of pairs of replicating and nonreplicating transcripts are shown in (A) that either failed to yield recombinants (NO RECOMBINATION) or yielded recombinants (RECOMBINATION) when Lipofectamine was used as the transfection reagent. Brackets indicate nonreplicating transcripts used individually in cotransfections with a common replicating transcript with a similar result. In some cases, transcript pairs that failed to yield recombinants following Lipofectaine-mediated transfection did yield recombinants when Lipofectamine 2000 was used, as indicated by an asterisk. To determine whether the J-UTR was a recombinational hot spot, DI-Stu and NR-5355-3′ transcripts generated from constructs that had been genetically tagged with restriction sites ( Nsi I at the 5′ end of the J-UTR or Xba I at the 3′ end of the J-UTR) were used in cotransfections (Lipofectamine 2000) and virus recovered from recombinant plaques was sequenced across the region of overlap in the middle of the genome. As shown in (B), recombinants were recovered that had no exchange of the parental markers, exchange of parental markers, and duplications of the 3′ NS-ORF and J-UTR.

    Techniques Used: Transfection, Generated, Construct, Recombinant

    Recombination at the 3′ end of the genome. Genomic diagrams of pairs of replicating and nonreplicating transcripts are shown in (A) that either failed to yield recombinants (NO RECOMBINATION) or yielded recombinants (RECOMBINATION) when Lipofectamine was used as the transfection reagent. Brackets indicate both replicating and nonreplicating transcripts used individually in cotransfections with a common replicating transcript(s) with a similar result. In some cases, transcript pairs that failed to yield recombinants following Lipofectaine-mediated transfection did yield recombinants when Lipofectamine 2000 was used, as indicated by an asterisk. In (B) is shown a model based on observations reported in (A) and previous reports that initiation of negative-strand synthesis is preferential for the NS-ORF and 3′CSE in cis . When one of the transcripts has the NS-ORF and 3′CSE in cis (i), negative-strand initiation and subsequent recombination is efficient to the extent that recombination is observed when Lipofectamine is used as the transfection reagent. When the NS-ORF and 3′CSE are in trans (iv), negative-strand initiation and recombination are less efficient and recombination is only observed when Lipofectamine 2000 is employed. This model predicts that when the NS-ORF and 3′CSE are available either in cis or in trans (ii and iii), initiation of minus-strand synthesis will occur preferentially on the 3′CSE in cis with the NS-ORF. To determine from which transcript the 3′ end of the recombinant was derived in such a situation, DI-Stu and NR-5355-3′ transcripts generated from constructs with a wt 3′ end or a construct with a deletion of 5 nt from the 3′ end of the genome [these constructs maintained the poly(A) tract] were used in cotransfections (Lipofectamine 2000) and virus recovered from recombinant plaques was sequenced across the region of overlap at the 3′ end of the genome. As shown in (C), recombinants uniformly had the 3′ end derived from the replicating parental transcript (DI-Stu).
    Figure Legend Snippet: Recombination at the 3′ end of the genome. Genomic diagrams of pairs of replicating and nonreplicating transcripts are shown in (A) that either failed to yield recombinants (NO RECOMBINATION) or yielded recombinants (RECOMBINATION) when Lipofectamine was used as the transfection reagent. Brackets indicate both replicating and nonreplicating transcripts used individually in cotransfections with a common replicating transcript(s) with a similar result. In some cases, transcript pairs that failed to yield recombinants following Lipofectaine-mediated transfection did yield recombinants when Lipofectamine 2000 was used, as indicated by an asterisk. In (B) is shown a model based on observations reported in (A) and previous reports that initiation of negative-strand synthesis is preferential for the NS-ORF and 3′CSE in cis . When one of the transcripts has the NS-ORF and 3′CSE in cis (i), negative-strand initiation and subsequent recombination is efficient to the extent that recombination is observed when Lipofectamine is used as the transfection reagent. When the NS-ORF and 3′CSE are in trans (iv), negative-strand initiation and recombination are less efficient and recombination is only observed when Lipofectamine 2000 is employed. This model predicts that when the NS-ORF and 3′CSE are available either in cis or in trans (ii and iii), initiation of minus-strand synthesis will occur preferentially on the 3′CSE in cis with the NS-ORF. To determine from which transcript the 3′ end of the recombinant was derived in such a situation, DI-Stu and NR-5355-3′ transcripts generated from constructs with a wt 3′ end or a construct with a deletion of 5 nt from the 3′ end of the genome [these constructs maintained the poly(A) tract] were used in cotransfections (Lipofectamine 2000) and virus recovered from recombinant plaques was sequenced across the region of overlap at the 3′ end of the genome. As shown in (C), recombinants uniformly had the 3′ end derived from the replicating parental transcript (DI-Stu).

    Techniques Used: Transfection, Recombinant, Derivative Assay, Generated, Construct

    Initial transcript pairs that yielded recombinants. At the top of (A) is a diagram of the RUB genome (ORFs as boxes, UTRs as lines); the vertical line in the J-UTR represents the SG RNA start site. Recombination was initially observed when the four replicating transcripts shown with deletions in the SP-ORF (the nt number in the RUB genome of the deletion breakpoints along with restriction sites used to generate the deletions are given;deletions are indicated by dotted lines) were individually used in cotransfections with the nonreplicating transcript NR-5355-3′. Virus from nine recombinant plaques generated from DI-Stu X NR-5355-3′ cotransfections were amplified in Vero cells. The amplified stocks were used to infect Vero cells and intracellular RNA was extracted and Northern analysis was used to characterize the viral RNAs present as shown in (B). The positions of migration of Robo402, DI-Stu, and NR-5355-3′ transcripts electrophoresed in the same gel are shown in the left margin, while the genome (G), subgenome (SG), and  28 S rRNA (which results in a white space) are indicated in the right margin. The RNA of five of these recombinants (2, 3, 4, 5, and 6) was sequenced across the region of overlap between the parental transcripts; the sequence of three of these recombinants was wild-type, while the other two recombinants had duplications in the 3′ end of the NS-ORF and J-UTR, as shown at the bottom (A). To test the effect of transfection reagent on recovery of recombinant virus, seven plates of Vero cells were cotransfected with DI-Stu and NR-5355-3′ transcripts using either Lipofectamine or Lipofectamine 2000. On days 4–10 posttransfection, the medium from one plate of cells was harvested and the virus present was titered by plaque assay; daily recovery of plaque-forming virus is shown in (C).
    Figure Legend Snippet: Initial transcript pairs that yielded recombinants. At the top of (A) is a diagram of the RUB genome (ORFs as boxes, UTRs as lines); the vertical line in the J-UTR represents the SG RNA start site. Recombination was initially observed when the four replicating transcripts shown with deletions in the SP-ORF (the nt number in the RUB genome of the deletion breakpoints along with restriction sites used to generate the deletions are given;deletions are indicated by dotted lines) were individually used in cotransfections with the nonreplicating transcript NR-5355-3′. Virus from nine recombinant plaques generated from DI-Stu X NR-5355-3′ cotransfections were amplified in Vero cells. The amplified stocks were used to infect Vero cells and intracellular RNA was extracted and Northern analysis was used to characterize the viral RNAs present as shown in (B). The positions of migration of Robo402, DI-Stu, and NR-5355-3′ transcripts electrophoresed in the same gel are shown in the left margin, while the genome (G), subgenome (SG), and 28 S rRNA (which results in a white space) are indicated in the right margin. The RNA of five of these recombinants (2, 3, 4, 5, and 6) was sequenced across the region of overlap between the parental transcripts; the sequence of three of these recombinants was wild-type, while the other two recombinants had duplications in the 3′ end of the NS-ORF and J-UTR, as shown at the bottom (A). To test the effect of transfection reagent on recovery of recombinant virus, seven plates of Vero cells were cotransfected with DI-Stu and NR-5355-3′ transcripts using either Lipofectamine or Lipofectamine 2000. On days 4–10 posttransfection, the medium from one plate of cells was harvested and the virus present was titered by plaque assay; daily recovery of plaque-forming virus is shown in (C).

    Techniques Used: Recombinant, Generated, Amplification, Northern Blot, Migration, Sequencing, Transfection, Plaque Assay

    17) Product Images from "Identification and characterization of new isoforms of human fas apoptotic inhibitory molecule (FAIM)"

    Article Title: Identification and characterization of new isoforms of human fas apoptotic inhibitory molecule (FAIM)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0185327

    Overexpression of nSR100 induce FAIM-L_2a and FAIM-L in HEK293T cells. A:  nSR100 transcript was amplified by RT-PCR. In SH-SY5Y cells, a band of 420 bp was detected.  B:  RT-PCR in HEK293T cells after transient transfection with nSR100 using Lipofectamine 2000®. Transcripts of FAIM-L and FAIM-L_2a were observed at 732 bp and 657 bp bands. L27 was used as an internal control in all PCRs.  C:  Western blot analysis using anti-FAIM in HEK293T cells after transfection with nSR100 vector (pLD_hsnSR100). A band of 23 kDa (FAIM-L) was detected in nSR100 transfection conditions. As a negative control, we used an empty vector (n = 3).
    Figure Legend Snippet: Overexpression of nSR100 induce FAIM-L_2a and FAIM-L in HEK293T cells. A: nSR100 transcript was amplified by RT-PCR. In SH-SY5Y cells, a band of 420 bp was detected. B: RT-PCR in HEK293T cells after transient transfection with nSR100 using Lipofectamine 2000®. Transcripts of FAIM-L and FAIM-L_2a were observed at 732 bp and 657 bp bands. L27 was used as an internal control in all PCRs. C: Western blot analysis using anti-FAIM in HEK293T cells after transfection with nSR100 vector (pLD_hsnSR100). A band of 23 kDa (FAIM-L) was detected in nSR100 transfection conditions. As a negative control, we used an empty vector (n = 3).

    Techniques Used: Over Expression, Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection, Western Blot, Plasmid Preparation, Negative Control

    18) Product Images from "KDM5 histone demethylases repress immune response via suppression of STING"

    Article Title: KDM5 histone demethylases repress immune response via suppression of STING

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2006134

    KDM5 inhibition induces a robust interferon response in cancer cells with elevated levels of cytosolic DNA. (A) Immunostaining of dsDNA and the mitochondrial marker Hsp60 in MCF7 cells. Surface plots of Z-stack images generated with Huygens (left panel). qPCR detecting mtDNA copy number in cells with the indicated treatment (right panel). 10 μM of ddC was used. Scale bar, 10 μm. (B, C) RT-qPCR (panel B) and western blot (panel C) analyses of MCF7 cells with the indicated treatment. MCF7 cells were treated with 10 μM ddC and 1 μM KDM5-C70 for 6 days. Cells were refed every 2 days. (D) dsDNA and DAPI staining of MCF7 cells or SKBR3 cells digested with 50 μg/ml DNase I. Surface plots of Z-stack images generated with Huygens (left panel). Quantification of dsDNA intensity per cell using image J was shown in the right panel. Scale bar, 10 μm. (E) RT-qPCR analyses of SKBR3 cells with the indicated treatment. SKBR3 cells were transfected with 1 μg dsDNA of about 90 bp (dsDNA90) using Lipofectamine 2000 5 hours before treatment with DMSO or 1 μM KDM5-C70 for 3 days. (F) RT-qPCR analysis of SKBR3 cells with knockout of the indicated genes after treatment with 1 μg dsDNA and 1 μM KDM5-C70 for 3 days. Representative data from triplicate experiments are shown. Error bar denotes SEM. # p
    Figure Legend Snippet: KDM5 inhibition induces a robust interferon response in cancer cells with elevated levels of cytosolic DNA. (A) Immunostaining of dsDNA and the mitochondrial marker Hsp60 in MCF7 cells. Surface plots of Z-stack images generated with Huygens (left panel). qPCR detecting mtDNA copy number in cells with the indicated treatment (right panel). 10 μM of ddC was used. Scale bar, 10 μm. (B, C) RT-qPCR (panel B) and western blot (panel C) analyses of MCF7 cells with the indicated treatment. MCF7 cells were treated with 10 μM ddC and 1 μM KDM5-C70 for 6 days. Cells were refed every 2 days. (D) dsDNA and DAPI staining of MCF7 cells or SKBR3 cells digested with 50 μg/ml DNase I. Surface plots of Z-stack images generated with Huygens (left panel). Quantification of dsDNA intensity per cell using image J was shown in the right panel. Scale bar, 10 μm. (E) RT-qPCR analyses of SKBR3 cells with the indicated treatment. SKBR3 cells were transfected with 1 μg dsDNA of about 90 bp (dsDNA90) using Lipofectamine 2000 5 hours before treatment with DMSO or 1 μM KDM5-C70 for 3 days. (F) RT-qPCR analysis of SKBR3 cells with knockout of the indicated genes after treatment with 1 μg dsDNA and 1 μM KDM5-C70 for 3 days. Representative data from triplicate experiments are shown. Error bar denotes SEM. # p

    Techniques Used: Inhibition, Immunostaining, Marker, Generated, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Staining, Transfection, Knock-Out

    19) Product Images from "Melatonin promotes the proliferation of GC-1 spg cells by inducing metallothionein-2 expression through ERK1/2 signaling pathway activation"

    Article Title: Melatonin promotes the proliferation of GC-1 spg cells by inducing metallothionein-2 expression through ERK1/2 signaling pathway activation

    Journal: Oncotarget

    doi: 10.18632/oncotarget.20019

    The cell proliferation assay after  Mt2  overexpression in GC-1 spg cells Overexpression plasmid of  Mt2  was constructed using pCMV6 plasmid. Plasmid was transfected into GC-1 spg cells with Lipofectamine 2000. Both mRNA ( A ) and protein ( B ) of  Mt2  were increased in cells with  Mt2  overexpression. Furthermore, cell proliferation assay showed that  Mt2  overexpression promoted cell proliferation and the expression of genes associated with cell proliferation ( C, D ). ★ indicates significant differences ( p
    Figure Legend Snippet: The cell proliferation assay after Mt2 overexpression in GC-1 spg cells Overexpression plasmid of Mt2 was constructed using pCMV6 plasmid. Plasmid was transfected into GC-1 spg cells with Lipofectamine 2000. Both mRNA ( A ) and protein ( B ) of Mt2 were increased in cells with Mt2 overexpression. Furthermore, cell proliferation assay showed that Mt2 overexpression promoted cell proliferation and the expression of genes associated with cell proliferation ( C, D ). ★ indicates significant differences ( p

    Techniques Used: Proliferation Assay, Over Expression, Plasmid Preparation, Construct, Transfection, Expressing

    20) Product Images from "Silencing Heat Shock Protein 27 (Hsp27) decreases metastatic behavior of human head and neck squamous cell cancer cells in vitro"

    Article Title: Silencing Heat Shock Protein 27 (Hsp27) decreases metastatic behavior of human head and neck squamous cell cancer cells in vitro

    Journal: Molecular pharmaceutics

    doi: 10.1021/mp100073s

    A. Microscopy image (×100) of cell invasion in UM-SCC-22B after siRNA transfection in one representative experiment. Ctrl, cells treated with transfection reagents lipofectamine 2000; Sictrl: cells transfected with scrambled siRNA, SiHsp27, cells
    Figure Legend Snippet: A. Microscopy image (×100) of cell invasion in UM-SCC-22B after siRNA transfection in one representative experiment. Ctrl, cells treated with transfection reagents lipofectamine 2000; Sictrl: cells transfected with scrambled siRNA, SiHsp27, cells

    Techniques Used: Microscopy, Transfection

    A. Real-time PCR to detect Hsp27 mRNA level in UM-SCC-22B cells after siRNA transfection. SiHsp27, transfected with siRNA of Hsp27. Ctrl, cell treated with transfection reagents lipofectamine 2000; Sictrl: cells transfected with scrambled siRNA. B. Western
    Figure Legend Snippet: A. Real-time PCR to detect Hsp27 mRNA level in UM-SCC-22B cells after siRNA transfection. SiHsp27, transfected with siRNA of Hsp27. Ctrl, cell treated with transfection reagents lipofectamine 2000; Sictrl: cells transfected with scrambled siRNA. B. Western

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection, Western Blot

    21) Product Images from "Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide"

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033287

    Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in   Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0).  P  values were calculated using a Student's  t -test. *,  P
    Figure Legend Snippet: Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0). P values were calculated using a Student's t -test. *, P

    Techniques Used: Migration, Transfection, Isolation

    A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).
    Figure Legend Snippet: A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Software, Isolation, Polymerase Chain Reaction, Luciferase, Negative Control, Incubation, Concentration Assay, Western Blot, Construct

    22) Product Images from "Angiotensin II-mediated posttranslational modification of nNOS in the PVN of rats with CHF: role for PIN"

    Article Title: Angiotensin II-mediated posttranslational modification of nNOS in the PVN of rats with CHF: role for PIN

    Journal: American Journal of Physiology - Heart and Circulatory Physiology

    doi: 10.1152/ajpheart.00170.2013

    PIN decreases the catalytically active form of nNOS in NG108 neurons. A : knockdown of PIN in NG108 cell line using siRNA. B : estimation of monomers and dimers of nNOS in NG108 cells transfected with Lipofectamine alone (Mock) or plasmid constructs: psi-PIN-shRNA
    Figure Legend Snippet: PIN decreases the catalytically active form of nNOS in NG108 neurons. A : knockdown of PIN in NG108 cell line using siRNA. B : estimation of monomers and dimers of nNOS in NG108 cells transfected with Lipofectamine alone (Mock) or plasmid constructs: psi-PIN-shRNA

    Techniques Used: Transfection, Plasmid Preparation, Construct, shRNA

    23) Product Images from "Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs"

    Article Title: Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl532

    Cells were transiently transfected with vector alone, a vector expressing 250 bp of luciferase dsRNA or a vector expressing 121 bp of c-met dsRNA using Lipofectamine 2000. Forty-eight hours after transfection, total RNA was isolated using Trizol and levels of c-met were quantified. The levels of c-met RNA were expressed relative to those found in vector alone transfected cells. * P
    Figure Legend Snippet: Cells were transiently transfected with vector alone, a vector expressing 250 bp of luciferase dsRNA or a vector expressing 121 bp of c-met dsRNA using Lipofectamine 2000. Forty-eight hours after transfection, total RNA was isolated using Trizol and levels of c-met were quantified. The levels of c-met RNA were expressed relative to those found in vector alone transfected cells. * P

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Luciferase, Isolation

    MDA-MB-231 cells were transiently transfected with either a plasmid encoding c-met long dsRNA or c-met siRNA by Lipofectamine 2000. Twenty-four hours after transfection, cells were added to a transwell chamber coated with matrigel, allowed to migrate for 48 h and the number of cells from the lower compartment of the chamber was compared with those transfected with vector alone. * P
    Figure Legend Snippet: MDA-MB-231 cells were transiently transfected with either a plasmid encoding c-met long dsRNA or c-met siRNA by Lipofectamine 2000. Twenty-four hours after transfection, cells were added to a transwell chamber coated with matrigel, allowed to migrate for 48 h and the number of cells from the lower compartment of the chamber was compared with those transfected with vector alone. * P

    Techniques Used: Multiple Displacement Amplification, Transfection, Plasmid Preparation

    Interferon β production at 48 h after transfection in the supernatant of MDA-MB-231 FFLuc cells transfected with lipofectamine or various dsRNA expression vectors or infected with adenovirus for 48 h. Interferon β levels in the supernatant were measured by ELISA and the IU/ml determined from a standard curve using recombinant interferon β.
    Figure Legend Snippet: Interferon β production at 48 h after transfection in the supernatant of MDA-MB-231 FFLuc cells transfected with lipofectamine or various dsRNA expression vectors or infected with adenovirus for 48 h. Interferon β levels in the supernatant were measured by ELISA and the IU/ml determined from a standard curve using recombinant interferon β.

    Techniques Used: Transfection, Multiple Displacement Amplification, Expressing, Infection, Enzyme-linked Immunosorbent Assay, Recombinant

    24) Product Images from "Effect of the miR-96-5p inhibitor and mimic on the migration and invasion of the SW480-7 colorectal cancer cell line"

    Article Title: Effect of the miR-96-5p inhibitor and mimic on the migration and invasion of the SW480-7 colorectal cancer cell line

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.10492

    Effects of miRNA-96-5p inhibitor on cell viability of (A) SW480, (B) SW620 and (C) HCT116. The values of cell viability were normalized with that of the corresponding negative control group. Untreated, only medium treated cells; LF, only Lipofectamine 2000 with medium treated cells; HiPerFect, only HiPerFect with medium treated cells; negative control, transfection with miR inhibitor control and cultured with medium; miR-96-5p, transfection with miR-96 inhibitor and cultured with medium. Data represent the mean ± SD of three independent experiments, each performed in triplicate.
    Figure Legend Snippet: Effects of miRNA-96-5p inhibitor on cell viability of (A) SW480, (B) SW620 and (C) HCT116. The values of cell viability were normalized with that of the corresponding negative control group. Untreated, only medium treated cells; LF, only Lipofectamine 2000 with medium treated cells; HiPerFect, only HiPerFect with medium treated cells; negative control, transfection with miR inhibitor control and cultured with medium; miR-96-5p, transfection with miR-96 inhibitor and cultured with medium. Data represent the mean ± SD of three independent experiments, each performed in triplicate.

    Techniques Used: Negative Control, Transfection, Cell Culture

    25) Product Images from "Tau reduction in the presence of amyloid-β prevents tau pathology and neuronal death in vivo"

    Article Title: Tau reduction in the presence of amyloid-β prevents tau pathology and neuronal death in vivo

    Journal: Brain

    doi: 10.1093/brain/awy117

    Human tau reduction decreases tau seeding activity despite amyloid-β presence. ( A ) A total of 0.1ug of total brain lysate per well was added to the HEK-TauRD CFP/YFP tau bioactivity sensor cells with Lipofectamine® and one well collected every 3 h for 24 h to measure the amount of aggregation—read out as FRET activity—over time for each sample. Integrated FRET Density was calculated for all samples by multiplying the per cent FRET positive cells by the median fluorescence intensity of the FRET positive population. Both the rTg4510 DOX (blue asterisk) and APP × rTg4510 DOX (green asterisk) samples exhibited less tau seeding activity than their naïve genotype controls. At the final 24 h point, a significant difference between the rTg4510 DOX and APP × rTg4510 DOX groups emerged (black asterisk). Treatment effect F (3,29) = 5.961, P = 0.0027. ( B ) Representative images of non-transduced cells (−), non-transgenic (NT), and APP/PS1 (APP) treated cells at the final 24 h collection point, all showing no aggregation. ( C ) Representative images of both rTg4510 and APP × rTg4510 genotypes and each treatment across all time points collected. Two-way repeated measures ANOVA, Sidak post hoc multiple comparisons. * P
    Figure Legend Snippet: Human tau reduction decreases tau seeding activity despite amyloid-β presence. ( A ) A total of 0.1ug of total brain lysate per well was added to the HEK-TauRD CFP/YFP tau bioactivity sensor cells with Lipofectamine® and one well collected every 3 h for 24 h to measure the amount of aggregation—read out as FRET activity—over time for each sample. Integrated FRET Density was calculated for all samples by multiplying the per cent FRET positive cells by the median fluorescence intensity of the FRET positive population. Both the rTg4510 DOX (blue asterisk) and APP × rTg4510 DOX (green asterisk) samples exhibited less tau seeding activity than their naïve genotype controls. At the final 24 h point, a significant difference between the rTg4510 DOX and APP × rTg4510 DOX groups emerged (black asterisk). Treatment effect F (3,29) = 5.961, P = 0.0027. ( B ) Representative images of non-transduced cells (−), non-transgenic (NT), and APP/PS1 (APP) treated cells at the final 24 h collection point, all showing no aggregation. ( C ) Representative images of both rTg4510 and APP × rTg4510 genotypes and each treatment across all time points collected. Two-way repeated measures ANOVA, Sidak post hoc multiple comparisons. * P

    Techniques Used: Activity Assay, Fluorescence, Transgenic Assay

    26) Product Images from "INFLAMMATION, CAVEOLAE AND CD38-MEDIATED CALCIUM REGULATION IN HUMAN AIRWAY SMOOTH MUSCLE"

    Article Title: INFLAMMATION, CAVEOLAE AND CD38-MEDIATED CALCIUM REGULATION IN HUMAN AIRWAY SMOOTH MUSCLE

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamcr.2013.11.011

    Effect of caveolin-1, cavin-1 and cavin-3 siRNA on intracellular Ca 2+ ([Ca 2+ ] i ) responses to agonist in human ASM cells. Compared to control cells (non-transfected, treated with Lipofectamine vehicle only), caveolin-1, cavin-1 and cavin-3 siRNA treated cells showed significantly reduced [Ca 2+ ] i responses to 10 μM histamine (A and B). TNFα significantly enhanced [Ca 2+ ] i responses to histamine. In the presence of TNFα, all caveolin-1 siRNA, cavin-1 siRNA and cavin-3 siRNA treated cells still showed significantly smaller [Ca 2+ ] i responses to histamine (C and D). Values are mean ± SEM. * indicates significant siRNA effect, # indicates significant effect of TNFα (p
    Figure Legend Snippet: Effect of caveolin-1, cavin-1 and cavin-3 siRNA on intracellular Ca 2+ ([Ca 2+ ] i ) responses to agonist in human ASM cells. Compared to control cells (non-transfected, treated with Lipofectamine vehicle only), caveolin-1, cavin-1 and cavin-3 siRNA treated cells showed significantly reduced [Ca 2+ ] i responses to 10 μM histamine (A and B). TNFα significantly enhanced [Ca 2+ ] i responses to histamine. In the presence of TNFα, all caveolin-1 siRNA, cavin-1 siRNA and cavin-3 siRNA treated cells still showed significantly smaller [Ca 2+ ] i responses to histamine (C and D). Values are mean ± SEM. * indicates significant siRNA effect, # indicates significant effect of TNFα (p

    Techniques Used: Transfection

    27) Product Images from "Angiogenin cleaves tRNA and promotes stress-induced translational repression"

    Article Title: Angiogenin cleaves tRNA and promotes stress-induced translational repression

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200811106

    Effect of endogenous tiRNAs on protein synthesis. (A) Endogenous 5′ and 3′ (lane 5), 5′ (lane 6), or 3′ (lane 7) tiRNAs extracted from angiogenin-treated U2OS cells were transfected into U2OS cells using lipofectamine. After 6 h, cells were pulsed with [ 35 S]methionine-containing medium for 30 min before protein extraction. Total counts per minute (cpm) per microgram of protein was normalized to cells treated with a combination of three PIWI-associated control RNAs (Control 1-2-3, lane 1; Control-1, lane 2; Control 3, lane 3) and expressed as a mean ± SD ( n = 3). *, P = 0.01; **, P = 0.01. (B) U2OS cells were transfected with the indicated control RNAs (lanes 1–5) or endogenous 5′ (lane 7), 3′ (lane 8), or 5′ and 3′ (lane 6) tiRNAs. After 6 h, cells were washed, and Trizol extracts were separated on a 15% TBD-urea gel and stained with CYBR gold. (C) Wild-type (SS) and S51A mutant (AA) MEFs were transfected with control RNAs (lanes 1 and 4) or endogenous 5′ (lanes 2 and 5) or 3′ (lanes 3 and 6) tiRNAs, pulsed with [ 35 S]methionine-containing medium, and extracted. Total cpm per microgram of protein was normalized to that of cells treated with control RNA and expressed as means ± SD ( n = 3). *, P = 0.01; **, P = 0.003. (D) Samples from C were separated by 15% SDS-PAGE, transferred to nitrocellulose, and exposed for autoradiography. Migration of molecular size markers is shown at the right.
    Figure Legend Snippet: Effect of endogenous tiRNAs on protein synthesis. (A) Endogenous 5′ and 3′ (lane 5), 5′ (lane 6), or 3′ (lane 7) tiRNAs extracted from angiogenin-treated U2OS cells were transfected into U2OS cells using lipofectamine. After 6 h, cells were pulsed with [ 35 S]methionine-containing medium for 30 min before protein extraction. Total counts per minute (cpm) per microgram of protein was normalized to cells treated with a combination of three PIWI-associated control RNAs (Control 1-2-3, lane 1; Control-1, lane 2; Control 3, lane 3) and expressed as a mean ± SD ( n = 3). *, P = 0.01; **, P = 0.01. (B) U2OS cells were transfected with the indicated control RNAs (lanes 1–5) or endogenous 5′ (lane 7), 3′ (lane 8), or 5′ and 3′ (lane 6) tiRNAs. After 6 h, cells were washed, and Trizol extracts were separated on a 15% TBD-urea gel and stained with CYBR gold. (C) Wild-type (SS) and S51A mutant (AA) MEFs were transfected with control RNAs (lanes 1 and 4) or endogenous 5′ (lanes 2 and 5) or 3′ (lanes 3 and 6) tiRNAs, pulsed with [ 35 S]methionine-containing medium, and extracted. Total cpm per microgram of protein was normalized to that of cells treated with control RNA and expressed as means ± SD ( n = 3). *, P = 0.01; **, P = 0.003. (D) Samples from C were separated by 15% SDS-PAGE, transferred to nitrocellulose, and exposed for autoradiography. Migration of molecular size markers is shown at the right.

    Techniques Used: Transfection, Protein Extraction, Staining, Mutagenesis, SDS Page, Autoradiography, Migration

    28) Product Images from "Upregulation of microRNA-98 increases radiosensitivity in esophageal squamous cell carcinoma"

    Article Title: Upregulation of microRNA-98 increases radiosensitivity in esophageal squamous cell carcinoma

    Journal: Journal of Radiation Research

    doi: 10.1093/jrr/rrw068

    Upregulation of miRNA-98 led to increased radiation sensitivity in radioresistant ESCC cells. (A) EC9706R cells were transfected with a miRNA-98 precursor vector, anti-miR-98 or a control vector using Lipofectamine. miRNA-98 expression was verified by qRT-PCR; U6 was chosen as an internal control. Asterisk indicates statistical significance ( P
    Figure Legend Snippet: Upregulation of miRNA-98 led to increased radiation sensitivity in radioresistant ESCC cells. (A) EC9706R cells were transfected with a miRNA-98 precursor vector, anti-miR-98 or a control vector using Lipofectamine. miRNA-98 expression was verified by qRT-PCR; U6 was chosen as an internal control. Asterisk indicates statistical significance ( P

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Quantitative RT-PCR

    29) Product Images from "Nicotine Enhances the Antiapoptotic Function of Mcl-1 through Phosphorylation *"

    Article Title: Nicotine Enhances the Antiapoptotic Function of Mcl-1 through Phosphorylation *

    Journal: Molecular cancer research : MCR

    doi: 10.1158/1541-7786.MCR-09-0304

    Mcl-1 is essential for nicotine-induced chemoresistance in human lung cancer H1299 cells A , Mcl-1 siRNA (15nM) or control siRNA (15 nM) was transfected into H1299 cells using Lipofectamine 2000. After 24h, the levels of Mcl-1 expression were analyzed by Western blot.  B , H1299 cells expressing Mcl-1 siRNA or control siRNA were treated with cisplatin (40 μM) in the absence or presence of nicotine (1 μM) for 96h. Cell viability was determined by analyzing annexin-V binding on FACS. Data represent the mean ± S.D. of three separate determinations.
    Figure Legend Snippet: Mcl-1 is essential for nicotine-induced chemoresistance in human lung cancer H1299 cells A , Mcl-1 siRNA (15nM) or control siRNA (15 nM) was transfected into H1299 cells using Lipofectamine 2000. After 24h, the levels of Mcl-1 expression were analyzed by Western blot. B , H1299 cells expressing Mcl-1 siRNA or control siRNA were treated with cisplatin (40 μM) in the absence or presence of nicotine (1 μM) for 96h. Cell viability was determined by analyzing annexin-V binding on FACS. Data represent the mean ± S.D. of three separate determinations.

    Techniques Used: Transfection, Expressing, Western Blot, Binding Assay, FACS

    30) Product Images from "The RNA-binding Protein HuR Stabilizes Cytosolic Phospholipase A2α mRNA under Interleukin-1β Treatment in Non-small Cell Lung Cancer A549 Cells"

    Article Title: The RNA-binding Protein HuR Stabilizes Cytosolic Phospholipase A2α mRNA under Interleukin-1β Treatment in Non-small Cell Lung Cancer A549 Cells

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.263582

    Reduction of HuR expression inhibits IL-1β-induced cPLA 2 α mRNA and protein expression.  Control siRNA and HuR siRNA were transfected into A549 cells by Lipofectamine 2000. After transfection for 24 h, cells were cultured in serum-free medium for 18 h and then treated with or without IL-1β (5 ng/ml) for 3 h.  A , total protein was extracted for analysis of the expression of HuR, tubulin, and cPLA 2 α.  B , the expression level of HuR and cPLA 2 α protein was quantified by densitometry.  C , the cPLA 2 α mRNA expression level was measure and quantified by quantitative RT-PCR (**,  p
    Figure Legend Snippet: Reduction of HuR expression inhibits IL-1β-induced cPLA 2 α mRNA and protein expression. Control siRNA and HuR siRNA were transfected into A549 cells by Lipofectamine 2000. After transfection for 24 h, cells were cultured in serum-free medium for 18 h and then treated with or without IL-1β (5 ng/ml) for 3 h. A , total protein was extracted for analysis of the expression of HuR, tubulin, and cPLA 2 α. B , the expression level of HuR and cPLA 2 α protein was quantified by densitometry. C , the cPLA 2 α mRNA expression level was measure and quantified by quantitative RT-PCR (**, p

    Techniques Used: Expressing, Transfection, Cell Culture, Quantitative RT-PCR

    31) Product Images from "A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing"

    Article Title: A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081156

    Characterisation of mouse ES cells cultured in 2i media. Fluorescence micrographs of GFP-expression E14 mES cells, pre-transfection cultured in 2i or SCM, transfected with Lipofectamine 2000 and pmaxGFP (A). Luciferase reporter assay for Oct4 and Nanog promoter activation, as well as Tead-enhanced promoter activation, 24 hrs post-transfection in E14 cells cultured without LIF in serum-free SCM with or without PD0325901 (1 μM) and CH99021 (3 μM), SCM, or 2i media. Results are mean ± sd (n = 3) p(*)
    Figure Legend Snippet: Characterisation of mouse ES cells cultured in 2i media. Fluorescence micrographs of GFP-expression E14 mES cells, pre-transfection cultured in 2i or SCM, transfected with Lipofectamine 2000 and pmaxGFP (A). Luciferase reporter assay for Oct4 and Nanog promoter activation, as well as Tead-enhanced promoter activation, 24 hrs post-transfection in E14 cells cultured without LIF in serum-free SCM with or without PD0325901 (1 μM) and CH99021 (3 μM), SCM, or 2i media. Results are mean ± sd (n = 3) p(*)

    Techniques Used: Cell Culture, Fluorescence, Expressing, Transfection, Luciferase, Reporter Assay, Activation Assay

    32) Product Images from "Silibinin down-regulates FAT10 and modulate TNF-α/IFN-γ-induced chromosomal instability and apoptosis sensitivity"

    Article Title: Silibinin down-regulates FAT10 and modulate TNF-α/IFN-γ-induced chromosomal instability and apoptosis sensitivity

    Journal: Biology Open

    doi: 10.1242/bio.011189

    Silibinin inhibits TNF-α/IFN-γ (TI)-induced endogenous FAT10 expression through NF-κB signaling pathway.  (A) Seven predicted binding sites of NF-κB were found within 2.5 kb upstream of FAT10 promoter region by GenomatixMatInspector Version 8.0 ( http://www.genomatix.de ). (B) p65 plays a role in modulating FAT10 expression induced by TI. Cells were electroporated with control siRNA (ctr) or siRNAs against p65 (p65) and grown for 24 h. They were then cultured with or without TI for 8 h before harvest. Inhibition of NF-κB subunit p65 expression by its specific siRNA and FAT10 expression were confirmed by western blot. (C) Silibinin inhibits TI-induced FAT10 mRNA levels in a dose dependent manner. Real-time reverse transcription (RT)-PCR was performed to measure the levels of FAT10 mRNA. (D1) Silibinin inhibits TI-induced FAT10 promoter and (D2) Silibinin inhibits NF-κB activities. FAT10-promoter driven β-galactosidase reporter and the NF-κB-SEAP constructs were co-transfected into HepG2 cells using Lipofectamine 2000. 36 h after transfection, cells were treated with TNF-α (50 ng/ml) and IFN-γ (50 U/ml), in the presence or absence of 100 µM silibinin. Nine hours later, cells were harvested and FAT10-promoter driven β-galactosidase reporter and NF-κB driven SEAP activities were determined and normalized against GFP expression and total protein content. All data are shown as mean±s.e. (standard error). ** P
    Figure Legend Snippet: Silibinin inhibits TNF-α/IFN-γ (TI)-induced endogenous FAT10 expression through NF-κB signaling pathway. (A) Seven predicted binding sites of NF-κB were found within 2.5 kb upstream of FAT10 promoter region by GenomatixMatInspector Version 8.0 ( http://www.genomatix.de ). (B) p65 plays a role in modulating FAT10 expression induced by TI. Cells were electroporated with control siRNA (ctr) or siRNAs against p65 (p65) and grown for 24 h. They were then cultured with or without TI for 8 h before harvest. Inhibition of NF-κB subunit p65 expression by its specific siRNA and FAT10 expression were confirmed by western blot. (C) Silibinin inhibits TI-induced FAT10 mRNA levels in a dose dependent manner. Real-time reverse transcription (RT)-PCR was performed to measure the levels of FAT10 mRNA. (D1) Silibinin inhibits TI-induced FAT10 promoter and (D2) Silibinin inhibits NF-κB activities. FAT10-promoter driven β-galactosidase reporter and the NF-κB-SEAP constructs were co-transfected into HepG2 cells using Lipofectamine 2000. 36 h after transfection, cells were treated with TNF-α (50 ng/ml) and IFN-γ (50 U/ml), in the presence or absence of 100 µM silibinin. Nine hours later, cells were harvested and FAT10-promoter driven β-galactosidase reporter and NF-κB driven SEAP activities were determined and normalized against GFP expression and total protein content. All data are shown as mean±s.e. (standard error). ** P

    Techniques Used: Expressing, Binding Assay, Cell Culture, Inhibition, Western Blot, Reverse Transcription Polymerase Chain Reaction, Construct, Transfection

    33) Product Images from "Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer"

    Article Title: Delivery of DNAzyme targeting aurora kinase A to inhibit the proliferation and migration of human prostate cancer

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S90559

    Induction of apoptosis in PC-3 cells treated with DNAzyme transfection determined by flow cytometry. Notes: ( A ) No treatment; ( B ) treatment with N -Ac- l -Leu-PEI; ( C ) treatment with N -Ac- l -Leu-PEI/iDz; ( D ) treatment with Lipofectamine™2000/Dz; and ( E ) treatment with N -Ac- l -Leu-PEI/Dz. Abbreviations: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine; iDz, inactive DNAzyme, PI, propidium iodide; FITC, fluorescein isothiocyanate.
    Figure Legend Snippet: Induction of apoptosis in PC-3 cells treated with DNAzyme transfection determined by flow cytometry. Notes: ( A ) No treatment; ( B ) treatment with N -Ac- l -Leu-PEI; ( C ) treatment with N -Ac- l -Leu-PEI/iDz; ( D ) treatment with Lipofectamine™2000/Dz; and ( E ) treatment with N -Ac- l -Leu-PEI/Dz. Abbreviations: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine; iDz, inactive DNAzyme, PI, propidium iodide; FITC, fluorescein isothiocyanate.

    Techniques Used: Transfection, Flow Cytometry, Cytometry

    Wound healing assay ( A ) and quantitative wound size ( B ) after DNAzyme transfection. Notes: 1: No treatment; 2: Lipofectamine™2000/Dz; 3: N -Ac- l -Leu-PEI/Dz; 4: N -Ac- l -Leu-PEI/iDz; and 5: N -Ac- l -Leu-PEI. The data were expressed as mean value ± SD of three experiments, and significance was determined by a Student’s t -test (* P
    Figure Legend Snippet: Wound healing assay ( A ) and quantitative wound size ( B ) after DNAzyme transfection. Notes: 1: No treatment; 2: Lipofectamine™2000/Dz; 3: N -Ac- l -Leu-PEI/Dz; 4: N -Ac- l -Leu-PEI/iDz; and 5: N -Ac- l -Leu-PEI. The data were expressed as mean value ± SD of three experiments, and significance was determined by a Student’s t -test (* P

    Techniques Used: Wound Healing Assay, Transfection

    Induction of cell cycle arrest in PC-3 cells treated with DNAzyme transfection determined by flow cytometry. Notes: ( A ) No treatment; ( B ) treatment with N -Ac- l -Leu-PEI for 24 hours; ( C ) treatment with N -Ac- l -Leu-PEI/iDz for 24 hours; ( D ) treatment with Lipofectamine™2000/Dz (6 μg DNAzyme) for 48 hours; ( E – G ) treatment with N -Ac- l -Leu-PEI/Dz for 24 hours with a DNAzyme amount of 2 μg, 4 μg, and 6 μg, respectively; and ( H ) treatment with N -Ac- l -Leu-PEI/Dz with a DNAzyme amount of 6 μg for 48 hours. Abbreviations: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine; iDz, inactive DNAzyme.
    Figure Legend Snippet: Induction of cell cycle arrest in PC-3 cells treated with DNAzyme transfection determined by flow cytometry. Notes: ( A ) No treatment; ( B ) treatment with N -Ac- l -Leu-PEI for 24 hours; ( C ) treatment with N -Ac- l -Leu-PEI/iDz for 24 hours; ( D ) treatment with Lipofectamine™2000/Dz (6 μg DNAzyme) for 48 hours; ( E – G ) treatment with N -Ac- l -Leu-PEI/Dz for 24 hours with a DNAzyme amount of 2 μg, 4 μg, and 6 μg, respectively; and ( H ) treatment with N -Ac- l -Leu-PEI/Dz with a DNAzyme amount of 6 μg for 48 hours. Abbreviations: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine; iDz, inactive DNAzyme.

    Techniques Used: Transfection, Flow Cytometry, Cytometry

    Western blotting ( A ) and quantitative analysis ( B ) for the expression level of aurora kinase A and MMP-9 after DNAzyme transfection. Notes: 1: No treatment; 2: N -Ac- l -Leu-PEI/Dz; 3: Lipofectamine™2000/Dz; and 4: N -Ac- l -Leu-PEI/iDz. Abbreviations: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine; iDz, inactive DNAzyme; MMP-9, matrix metalloproteinase-9.
    Figure Legend Snippet: Western blotting ( A ) and quantitative analysis ( B ) for the expression level of aurora kinase A and MMP-9 after DNAzyme transfection. Notes: 1: No treatment; 2: N -Ac- l -Leu-PEI/Dz; 3: Lipofectamine™2000/Dz; and 4: N -Ac- l -Leu-PEI/iDz. Abbreviations: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine; iDz, inactive DNAzyme; MMP-9, matrix metalloproteinase-9.

    Techniques Used: Western Blot, Expressing, Transfection

    Effects of DNAzyme transfection on the cell migration using Transwell migration assay: ( A ) no treatment; ( B ) N -Ac- l -Leu-PEI; ( C ) N -Ac- l -Leu-PEI/iDz; ( D ) Lipofectamine™2000/Dz; and ( E ) N -Ac- l -Leu-PEI/Dz. Abbreviation: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine.
    Figure Legend Snippet: Effects of DNAzyme transfection on the cell migration using Transwell migration assay: ( A ) no treatment; ( B ) N -Ac- l -Leu-PEI; ( C ) N -Ac- l -Leu-PEI/iDz; ( D ) Lipofectamine™2000/Dz; and ( E ) N -Ac- l -Leu-PEI/Dz. Abbreviation: N -Ac- l -Leu-PEI, N -acetyl- l -leucine-polyethylenimine.

    Techniques Used: Transfection, Migration, Transwell Migration Assay

    34) Product Images from "Innovative DNA Vaccine for Human Papillomavirus (HPV)-Associated Head and Neck Cancer"

    Article Title: Innovative DNA Vaccine for Human Papillomavirus (HPV)-Associated Head and Neck Cancer

    Journal: Gene therapy

    doi: 10.1038/gt.2010.151

    Characterization of the activation of the E6-specific T cell line by 293-K b  cells transfected with the various DNA constructs 293 cells expressing murine H-2K b  (293-K b ) were transfected with pcDNA3 encoding Ii, PADRE, E6, Ii-E6, and PADRE-E6 respectively using Lipofectamine 2000. 24 hours later, transfected cells were used to stimulate a H-2K b  restricted, HPV-16 E6 aa48–57 specific T cell line for 20 hours at the presence of 1μl/ml of GolgiPlug. 293-K b  cells pulsed with HPV16 E6aa50–57 peptides were used as positive control. The cells were then analyzed using intracellular cytokine staining by staining with CD8 and IFN-γ.  A.  Representative flow cytometry data demonstrating the percentage of IFN-γ +  CD8 +  T cells out of total CD8 +  T cells.  B.  Bar graph depicting the percentage of IFN-γ +  CD8 +  T cells out of total CD8 +  T cells. The data shown here are from one representative experiment of two performed.
    Figure Legend Snippet: Characterization of the activation of the E6-specific T cell line by 293-K b cells transfected with the various DNA constructs 293 cells expressing murine H-2K b (293-K b ) were transfected with pcDNA3 encoding Ii, PADRE, E6, Ii-E6, and PADRE-E6 respectively using Lipofectamine 2000. 24 hours later, transfected cells were used to stimulate a H-2K b restricted, HPV-16 E6 aa48–57 specific T cell line for 20 hours at the presence of 1μl/ml of GolgiPlug. 293-K b cells pulsed with HPV16 E6aa50–57 peptides were used as positive control. The cells were then analyzed using intracellular cytokine staining by staining with CD8 and IFN-γ. A. Representative flow cytometry data demonstrating the percentage of IFN-γ + CD8 + T cells out of total CD8 + T cells. B. Bar graph depicting the percentage of IFN-γ + CD8 + T cells out of total CD8 + T cells. The data shown here are from one representative experiment of two performed.

    Techniques Used: Activation Assay, Transfection, Construct, Expressing, Positive Control, Staining, Flow Cytometry, Cytometry

    35) Product Images from "Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells"

    Article Title: Regulation of Akt/FoxO3a/Skp2 Axis Is Critically Involved in Berberine-Induced Cell Cycle Arrest in Hepatocellular Carcinoma Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020327

    BBR-induced G0/G1 phase cell cycle arrest is attributed to FoxO3a up-regulation in Huh-7 cells. ( A ) Knockdown of FoxO3a induces down-regulation of p21 Cip1 and p27 Kip1 , up-regulation of Skp2. Huh-7 cells were transfected with control and FoxO3a siRNA with lipofectamine 2000 for 48 h, followed by exposure the cells to berberine (120 μM) for an additional 24 h. The protein levels of FoxO3a, p21 Cip1 , p27 Kip1 and Skp2 were examined by western blot. Bar graphs demonstrate relative expression levels of indicated proteins. ## represents p
    Figure Legend Snippet: BBR-induced G0/G1 phase cell cycle arrest is attributed to FoxO3a up-regulation in Huh-7 cells. ( A ) Knockdown of FoxO3a induces down-regulation of p21 Cip1 and p27 Kip1 , up-regulation of Skp2. Huh-7 cells were transfected with control and FoxO3a siRNA with lipofectamine 2000 for 48 h, followed by exposure the cells to berberine (120 μM) for an additional 24 h. The protein levels of FoxO3a, p21 Cip1 , p27 Kip1 and Skp2 were examined by western blot. Bar graphs demonstrate relative expression levels of indicated proteins. ## represents p

    Techniques Used: Transfection, Western Blot, Expressing

    36) Product Images from "Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells"

    Article Title: Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-12-27

    Comparison of activation of Hspa1b promoter by different transfection agents (CAT assay) . The B16F10 cells were transfected with p950/CAT6 construct containing Hspa1b promoter linked with CAT reporter gene using: Lipofectin (Gibco), DEAE-Dextran (Pharmacia), DDAB/DOPE (home made) and Lipofectamine 2000 (Invitrogen). K - cells grown at 37°C for 6 or 48 h after transfection. HS - heat-shocked cells, grown at 37°C for 24 h after transfection, then heat shocked at 42,5°C for 45 min followed by recovery for 24 h at 37°C. AcCM - acetylchloramphenicol, product of the enzymatic assay. CM - non-acetylated chloramphenicol, substrate of the assay.
    Figure Legend Snippet: Comparison of activation of Hspa1b promoter by different transfection agents (CAT assay) . The B16F10 cells were transfected with p950/CAT6 construct containing Hspa1b promoter linked with CAT reporter gene using: Lipofectin (Gibco), DEAE-Dextran (Pharmacia), DDAB/DOPE (home made) and Lipofectamine 2000 (Invitrogen). K - cells grown at 37°C for 6 or 48 h after transfection. HS - heat-shocked cells, grown at 37°C for 24 h after transfection, then heat shocked at 42,5°C for 45 min followed by recovery for 24 h at 37°C. AcCM - acetylchloramphenicol, product of the enzymatic assay. CM - non-acetylated chloramphenicol, substrate of the assay.

    Techniques Used: Activation Assay, Transfection, Construct, Enzymatic Assay

    37) Product Images from "PEG-conjugated PAMAM Dendrimers Mediate Efficient Intramuscular Gene Expression"

    Article Title: PEG-conjugated PAMAM Dendrimers Mediate Efficient Intramuscular Gene Expression

    Journal: The AAPS Journal

    doi: 10.1208/s12248-009-9116-1

    Effect of different PEG conjugation ratios on the transfection efficacy of PEG-G5 and PEG-G6 on 293A cell, with Lipofectamine 2000 ( LF 2000 ) as a positive control and pEGFP as a model of DNA. Statistically significant differences are denoted by * ( P
    Figure Legend Snippet: Effect of different PEG conjugation ratios on the transfection efficacy of PEG-G5 and PEG-G6 on 293A cell, with Lipofectamine 2000 ( LF 2000 ) as a positive control and pEGFP as a model of DNA. Statistically significant differences are denoted by * ( P

    Techniques Used: Conjugation Assay, Transfection, Positive Control

    38) Product Images from "Effects of miR-21 downregulation and silibinin treatment in breast cancer cell lines"

    Article Title: Effects of miR-21 downregulation and silibinin treatment in breast cancer cell lines

    Journal: Cytotechnology

    doi: 10.1007/s10616-017-0076-5

    Transfection of breast cancer cell lines by fluorescein labeled-scrambled LNA. a Phase contrast microscopy, scale bars 100 μm, b fluorescent microscopy and c flow cytometric analysis of transfected cells, percentages show the transfection efficiencies. The green spots in the fluorescent pictures indicate the cytoplasmic lipofectamine containing fluorescent scrambled. (Color figure online)
    Figure Legend Snippet: Transfection of breast cancer cell lines by fluorescein labeled-scrambled LNA. a Phase contrast microscopy, scale bars 100 μm, b fluorescent microscopy and c flow cytometric analysis of transfected cells, percentages show the transfection efficiencies. The green spots in the fluorescent pictures indicate the cytoplasmic lipofectamine containing fluorescent scrambled. (Color figure online)

    Techniques Used: Transfection, Labeling, Microscopy, Flow Cytometry

    39) Product Images from "Aquaporin 1 contributes to chondrocyte apoptosis in a rat model of osteoarthritis"

    Article Title: Aquaporin 1 contributes to chondrocyte apoptosis in a rat model of osteoarthritis

    Journal: International Journal of Molecular Medicine

    doi: 10.3892/ijmm.2016.2785

    (A) Caspase-3 mRNA expression in control cells, cells treated with Lipofectamine ®  2000 alone, cells transfected with scrambled shRNA, and cells transfected with AQPl-1-pGenesil-1.  * P
    Figure Legend Snippet: (A) Caspase-3 mRNA expression in control cells, cells treated with Lipofectamine ® 2000 alone, cells transfected with scrambled shRNA, and cells transfected with AQPl-1-pGenesil-1. * P

    Techniques Used: Expressing, Transfection, shRNA

    Positive transfection rate detected by flow cytometry. (A) Transfected cartilage cells with (A) no GFP expression; (B) transfected cartilage cells with GFP expression. (C) AQP-1 mRNA expression in control cells, cells treated with Lipofectamine ®  2000 alone, cells transfected with scrambled shRNA, and cells transfected with AQPl-1-pGenesil-1.  * P
    Figure Legend Snippet: Positive transfection rate detected by flow cytometry. (A) Transfected cartilage cells with (A) no GFP expression; (B) transfected cartilage cells with GFP expression. (C) AQP-1 mRNA expression in control cells, cells treated with Lipofectamine ® 2000 alone, cells transfected with scrambled shRNA, and cells transfected with AQPl-1-pGenesil-1. * P

    Techniques Used: Transfection, Flow Cytometry, Cytometry, Expressing, shRNA

    40) Product Images from "Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication ▿"

    Article Title: Lack of Bax Prevents Influenza A Virus-Induced Apoptosis and Causes Diminished Viral Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02672-08

    Influenza A virus replication is dependent upon opposite virus-induced effects on Bax and Bak activity that are unlikely to be interferon related. (A) Influenza A virus replication was analyzed by plaque assay. Virus replication is severely attenuated in Bax KO cells, resulting in a 2-log decrease in PFU/ml compared to the WT. Bak KO cells allow a maximum replication similar to that of the WT, while Bax/Bak DKO cells show a slight elevation of infectious titers during infection. These results indicate that Bax is proviral during infection, while Bak is dispensable for replication. (B) Bax was transiently expressed in all cell types by Lipofectamine 2000 transfection of a C2-Bax-GFP construct prior to infection, and supernatant samples were collected for plaque assay at 48 hpi. Baseline virus replication in each cell type was evaluated using empty C2-GFP plasmid transfection. Bax reconstitution in Bax KO cells resulted in a fivefold increase in infectious titers compared to the control ( P = 0.0007). A minimal effect on the virus titer was seen after Bax overexpression by transient transfection in WT cells compared to empty plasmid controls. (C) Influenza A virus replication was assessed by reverse transcription-PCR. Serial dilutions of stock virus at known concentrations were also analyzed to generate a standard curve to which experimental samples were compared, thus calculating the approximate number of influenza A virus particles/ml in each sample. By 24 hpi, Bax KO, Bak KO, and Bax/Bak DKO cells all showed significantly higher levels of influenza A virus RNA released into the cell culture supernatant than did WT cells. (D) Interferon activity was assessed by infecting mock- and influenza A virus-infected cells with interferon-sensitive, GFP-linked NDV and quantifying the mean GFP expression levels of 10,000 events per condition by FACS analysis. Each assay was run in triplicate, and data are expressed as the ratio of the numbers of influenza A virus-infected to mock-infected cells per cell type. After influenza A virus infection, Bak KO cells exhibited a slight decrease in ratio compared to the WT, representing a 30% increase in interferon activity ( P = 0.002). Bax KO and Bax/Bak DKO cells both showed similar fluorescence changes compared to the WT after infection. Due the high degree of similarity between cell types, these results suggest that the interferon response in infected cells is modulated by viral replication in the presence of Bak and is only slightly modified by Bax activity during influenza A virus infection. As an elevated interferon response typically leads to a reduced virus replication capacity, these results also suggest that it is unlikely that the observed trends in infectious virus titer are due to virus-induced interferon signaling.
    Figure Legend Snippet: Influenza A virus replication is dependent upon opposite virus-induced effects on Bax and Bak activity that are unlikely to be interferon related. (A) Influenza A virus replication was analyzed by plaque assay. Virus replication is severely attenuated in Bax KO cells, resulting in a 2-log decrease in PFU/ml compared to the WT. Bak KO cells allow a maximum replication similar to that of the WT, while Bax/Bak DKO cells show a slight elevation of infectious titers during infection. These results indicate that Bax is proviral during infection, while Bak is dispensable for replication. (B) Bax was transiently expressed in all cell types by Lipofectamine 2000 transfection of a C2-Bax-GFP construct prior to infection, and supernatant samples were collected for plaque assay at 48 hpi. Baseline virus replication in each cell type was evaluated using empty C2-GFP plasmid transfection. Bax reconstitution in Bax KO cells resulted in a fivefold increase in infectious titers compared to the control ( P = 0.0007). A minimal effect on the virus titer was seen after Bax overexpression by transient transfection in WT cells compared to empty plasmid controls. (C) Influenza A virus replication was assessed by reverse transcription-PCR. Serial dilutions of stock virus at known concentrations were also analyzed to generate a standard curve to which experimental samples were compared, thus calculating the approximate number of influenza A virus particles/ml in each sample. By 24 hpi, Bax KO, Bak KO, and Bax/Bak DKO cells all showed significantly higher levels of influenza A virus RNA released into the cell culture supernatant than did WT cells. (D) Interferon activity was assessed by infecting mock- and influenza A virus-infected cells with interferon-sensitive, GFP-linked NDV and quantifying the mean GFP expression levels of 10,000 events per condition by FACS analysis. Each assay was run in triplicate, and data are expressed as the ratio of the numbers of influenza A virus-infected to mock-infected cells per cell type. After influenza A virus infection, Bak KO cells exhibited a slight decrease in ratio compared to the WT, representing a 30% increase in interferon activity ( P = 0.002). Bax KO and Bax/Bak DKO cells both showed similar fluorescence changes compared to the WT after infection. Due the high degree of similarity between cell types, these results suggest that the interferon response in infected cells is modulated by viral replication in the presence of Bak and is only slightly modified by Bax activity during influenza A virus infection. As an elevated interferon response typically leads to a reduced virus replication capacity, these results also suggest that it is unlikely that the observed trends in infectious virus titer are due to virus-induced interferon signaling.

    Techniques Used: Activity Assay, Plaque Assay, Infection, Transfection, Construct, Plasmid Preparation, Over Expression, Polymerase Chain Reaction, Cell Culture, Expressing, FACS, Fluorescence, Modification

    Related Articles

    MTT Assay:

    Article Title: Influence of Oligospermines Architecture on Their Suitability for siRNA Delivery
    Article Snippet: .. Lipofectamine 2000 (LF), SYBR Gold dye, and (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Life Technologies (Grand Island, NY). .. Polyethylenimine (PEI, MW 5 kDa) was obtained from BASF (Lupasol, Cologne, Germany).

    Transfection:

    Article Title: Knockdown of Dinoflagellate Cellulose Synthase CesA1 Resulted in Malformed Intracellular Cellulosic Thecal Plates and Severely Impeded Cyst-to-Swarmer Transition
    Article Snippet: .. For each transfection sample, oligomer-Lipofectamine 2000 complexes were prepared by mixing 300 μl L medium, 300 μl 12 nmole antisense or scrambled ODN and 30 μl Lipofectamine Reagent 2000 (Invitrogen). .. The mixture was incubated for 30 min at room temperature (with rotation), before incubating with 300 μl spheroplasts (original culture: 800 ml, final cell number: ∼1.8 × 106 ) for another 1 h at room temperature (with gentle rotation).

    Article Title: Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines
    Article Snippet: .. ERβ transcriptional activity assay BLM cells, seeded in 24-well plates (5x104 cells/well) in phenol red free DMEM medium supplemented with 10% charcoal stripped FBS, were transfected using Lipofectamine 2000 (Life Technologies, Monza, Italy), according to the manufacturer’s protocol [ ]. .. The following constructs were cotransfected: pVERE-tk-Luc (1μg), the reporter plasmid encoding the firefly luciferase reporter gene under the control of the estrogen response element (ERE; kindly provided by Dr. A. Maggi, Department of Pharmacological and Biomolecular Sciences, University of Milano, Milano, Italy), to evaluate the transcriptional activity of ERβ, and pCMVβ (0.4 μg), the reporter plasmid encoding the β-galactosidase (Clontech, Jesi, Italy), as the internal control plasmid.

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy
    Article Snippet: .. Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above. .. Unless otherwise stated, cells were transfected twice at 24 h intervals in 37 °C and 5% CO2 with 200 μL of micelleplex suspension containing 100 pmol siRNA within a total volume of 2 mL of serum-containing cell culture media.

    Article Title: Mitochondrial-targeted Aryl Hydrocarbon Receptor and the Impact of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Cellular Respiration and the Mitochondrial Proteome
    Article Snippet: .. When cells were 50% confluent, the siRNAs specific for AIP (Ambion s62179 (siAIP1), s62181 (siAIP2), Life Technologies), and the Silencer® Select Negative Control no. 1 siRNA (Ambion) were transfected into hepa1c1c7 cells using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. .. For TOMM20 knockdown, the siRNA for TOMM20 (Tomm20 ON-TARGET plus, SMARTpool L-006487-01-0005, Dharmacon, GE) and the nontargeting siRNA (Dharmacon, GE) were transfected into hepa1c1c7 cells.

    Recombinant:

    Article Title: Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate
    Article Snippet: .. Reagents were purchased as followings: recombinant human TNFα (rcyc-htnfs) from InvivoGene; cycloheximide (HY-12320), navitoclax (HY-10087), actinomycin D(HY-17559), broad spectrum caspase inhibitor Q-VD-OPh (HY-12305) from MedChemExpress; caspase-3 inhitor Q-DEVD-OPh (1175-1) from Biovision, protease inhibitor cocktail tablets (04693132001) from Roche; protein G sepharose (17-0618-01) from GE Healthcare; Lipofectamine 2000 (11668-019) and Lipofectamine RNAiMAX (13778-150) from Invitrogen; anti-Flag M2 magnetic beads (M8823) and 2-BP (238422) from Sigma-Aldrich; CytoTox 96 Non-Radio cytotoxicity assay kit (G1780) from Promega; Hoechst 33342/PI double stain kit (CA1120) from Solarbio; and FITC annexin V appotosis kit I (556547) from BD Biosciences. .. Cell culture and treatments The human colon cancer cell lines HCT116 wild type and BAK −/− BAX −/− (DKO), the mouse embryonic fibroblasts MEFs, the human ovarian cancer cell lines including Ovcar3, Ovcar5, Ovcar8, A2780, HeyA8, OV90, COV362, SKOV3, Ovise, and the hematoma cell lines including V937, MOLT-3, MOLT-4, THP-1, SKW6.4, H9, Jurkat, K562, KG1a, RL, ML-1, HL-60, HT, and MV4-11 were kind gifts from Scott H. Kaufmann (Mayo Clinic, Rochester, MN).

    Positive Control:

    Article Title: Nanoparticle-Mediated Gene Silencing for Sensitization of Lung Cancer to Cisplatin Therapy
    Article Snippet: .. Negative controls consisted of untreated cells or cells transfected with control scrambled siRNA while positive control cells were transfected with Lipofectamine 2000 (Life Technologies) lipoplexes, which were prepared as described above. .. Unless otherwise stated, cells were transfected twice at 24 h intervals in 37 °C and 5% CO2 with 200 μL of micelleplex suspension containing 100 pmol siRNA within a total volume of 2 mL of serum-containing cell culture media.

    Magnetic Beads:

    Article Title: Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate
    Article Snippet: .. Reagents were purchased as followings: recombinant human TNFα (rcyc-htnfs) from InvivoGene; cycloheximide (HY-12320), navitoclax (HY-10087), actinomycin D(HY-17559), broad spectrum caspase inhibitor Q-VD-OPh (HY-12305) from MedChemExpress; caspase-3 inhitor Q-DEVD-OPh (1175-1) from Biovision, protease inhibitor cocktail tablets (04693132001) from Roche; protein G sepharose (17-0618-01) from GE Healthcare; Lipofectamine 2000 (11668-019) and Lipofectamine RNAiMAX (13778-150) from Invitrogen; anti-Flag M2 magnetic beads (M8823) and 2-BP (238422) from Sigma-Aldrich; CytoTox 96 Non-Radio cytotoxicity assay kit (G1780) from Promega; Hoechst 33342/PI double stain kit (CA1120) from Solarbio; and FITC annexin V appotosis kit I (556547) from BD Biosciences. .. Cell culture and treatments The human colon cancer cell lines HCT116 wild type and BAK −/− BAX −/− (DKO), the mouse embryonic fibroblasts MEFs, the human ovarian cancer cell lines including Ovcar3, Ovcar5, Ovcar8, A2780, HeyA8, OV90, COV362, SKOV3, Ovise, and the hematoma cell lines including V937, MOLT-3, MOLT-4, THP-1, SKW6.4, H9, Jurkat, K562, KG1a, RL, ML-1, HL-60, HT, and MV4-11 were kind gifts from Scott H. Kaufmann (Mayo Clinic, Rochester, MN).

    Protease Inhibitor:

    Article Title: Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate
    Article Snippet: .. Reagents were purchased as followings: recombinant human TNFα (rcyc-htnfs) from InvivoGene; cycloheximide (HY-12320), navitoclax (HY-10087), actinomycin D(HY-17559), broad spectrum caspase inhibitor Q-VD-OPh (HY-12305) from MedChemExpress; caspase-3 inhitor Q-DEVD-OPh (1175-1) from Biovision, protease inhibitor cocktail tablets (04693132001) from Roche; protein G sepharose (17-0618-01) from GE Healthcare; Lipofectamine 2000 (11668-019) and Lipofectamine RNAiMAX (13778-150) from Invitrogen; anti-Flag M2 magnetic beads (M8823) and 2-BP (238422) from Sigma-Aldrich; CytoTox 96 Non-Radio cytotoxicity assay kit (G1780) from Promega; Hoechst 33342/PI double stain kit (CA1120) from Solarbio; and FITC annexin V appotosis kit I (556547) from BD Biosciences. .. Cell culture and treatments The human colon cancer cell lines HCT116 wild type and BAK −/− BAX −/− (DKO), the mouse embryonic fibroblasts MEFs, the human ovarian cancer cell lines including Ovcar3, Ovcar5, Ovcar8, A2780, HeyA8, OV90, COV362, SKOV3, Ovise, and the hematoma cell lines including V937, MOLT-3, MOLT-4, THP-1, SKW6.4, H9, Jurkat, K562, KG1a, RL, ML-1, HL-60, HT, and MV4-11 were kind gifts from Scott H. Kaufmann (Mayo Clinic, Rochester, MN).

    Negative Control:

    Article Title: Mitochondrial-targeted Aryl Hydrocarbon Receptor and the Impact of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin on Cellular Respiration and the Mitochondrial Proteome
    Article Snippet: .. When cells were 50% confluent, the siRNAs specific for AIP (Ambion s62179 (siAIP1), s62181 (siAIP2), Life Technologies), and the Silencer® Select Negative Control no. 1 siRNA (Ambion) were transfected into hepa1c1c7 cells using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. .. For TOMM20 knockdown, the siRNA for TOMM20 (Tomm20 ON-TARGET plus, SMARTpool L-006487-01-0005, Dharmacon, GE) and the nontargeting siRNA (Dharmacon, GE) were transfected into hepa1c1c7 cells.

    Mouse Assay:

    Article Title: Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene
    Article Snippet: .. In each experiment, four tumor-bearing or healthy mice were injected intravenously (i.v.), intraperitoneally (i.p.), intramuscularly (i.m.), subcutaneously (s.c.), or peritumorally (p.t.) with 1.7 μg/g Cy7 or Cy5.5-labeled siRNA, Ch-siRNA, or siRNA/Lipofectamine 2000 complex in 100 μL Opti-MEM medium (Invitrogen), and the fifth mouse was left intact as a control. ..

    Incubation:

    Article Title: Aβ and tau prion-like activities decline with longevity in Alzheimer’s disease brains
    Article Snippet: .. A mixture of Lipofectamine 2000 (1.5% final volume; Thermo Fisher), OptiMEM (78.5% final volume; Thermo Fisher) and sample (20% final volume) were incubated at room temperature for 2 hours and plated in four replicate wells (10 μL/well). .. Plates were then incubated and imaged every 24 hours on the IN Cell 6000 (GE Healthcare) for 3–4 days.

    Activity Assay:

    Article Title: Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines
    Article Snippet: .. ERβ transcriptional activity assay BLM cells, seeded in 24-well plates (5x104 cells/well) in phenol red free DMEM medium supplemented with 10% charcoal stripped FBS, were transfected using Lipofectamine 2000 (Life Technologies, Monza, Italy), according to the manufacturer’s protocol [ ]. .. The following constructs were cotransfected: pVERE-tk-Luc (1μg), the reporter plasmid encoding the firefly luciferase reporter gene under the control of the estrogen response element (ERE; kindly provided by Dr. A. Maggi, Department of Pharmacological and Biomolecular Sciences, University of Milano, Milano, Italy), to evaluate the transcriptional activity of ERβ, and pCMVβ (0.4 μg), the reporter plasmid encoding the β-galactosidase (Clontech, Jesi, Italy), as the internal control plasmid.

    Staining:

    Article Title: Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate
    Article Snippet: .. Reagents were purchased as followings: recombinant human TNFα (rcyc-htnfs) from InvivoGene; cycloheximide (HY-12320), navitoclax (HY-10087), actinomycin D(HY-17559), broad spectrum caspase inhibitor Q-VD-OPh (HY-12305) from MedChemExpress; caspase-3 inhitor Q-DEVD-OPh (1175-1) from Biovision, protease inhibitor cocktail tablets (04693132001) from Roche; protein G sepharose (17-0618-01) from GE Healthcare; Lipofectamine 2000 (11668-019) and Lipofectamine RNAiMAX (13778-150) from Invitrogen; anti-Flag M2 magnetic beads (M8823) and 2-BP (238422) from Sigma-Aldrich; CytoTox 96 Non-Radio cytotoxicity assay kit (G1780) from Promega; Hoechst 33342/PI double stain kit (CA1120) from Solarbio; and FITC annexin V appotosis kit I (556547) from BD Biosciences. .. Cell culture and treatments The human colon cancer cell lines HCT116 wild type and BAK −/− BAX −/− (DKO), the mouse embryonic fibroblasts MEFs, the human ovarian cancer cell lines including Ovcar3, Ovcar5, Ovcar8, A2780, HeyA8, OV90, COV362, SKOV3, Ovise, and the hematoma cell lines including V937, MOLT-3, MOLT-4, THP-1, SKW6.4, H9, Jurkat, K562, KG1a, RL, ML-1, HL-60, HT, and MV4-11 were kind gifts from Scott H. Kaufmann (Mayo Clinic, Rochester, MN).

    Injection:

    Article Title: Cholesterol-Containing Nuclease-Resistant siRNA Accumulates in Tumors in a Carrier-free Mode and Silences MDR1 Gene
    Article Snippet: .. In each experiment, four tumor-bearing or healthy mice were injected intravenously (i.v.), intraperitoneally (i.p.), intramuscularly (i.m.), subcutaneously (s.c.), or peritumorally (p.t.) with 1.7 μg/g Cy7 or Cy5.5-labeled siRNA, Ch-siRNA, or siRNA/Lipofectamine 2000 complex in 100 μL Opti-MEM medium (Invitrogen), and the fifth mouse was left intact as a control. ..

    Cytotoxicity Assay:

    Article Title: Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate
    Article Snippet: .. Reagents were purchased as followings: recombinant human TNFα (rcyc-htnfs) from InvivoGene; cycloheximide (HY-12320), navitoclax (HY-10087), actinomycin D(HY-17559), broad spectrum caspase inhibitor Q-VD-OPh (HY-12305) from MedChemExpress; caspase-3 inhitor Q-DEVD-OPh (1175-1) from Biovision, protease inhibitor cocktail tablets (04693132001) from Roche; protein G sepharose (17-0618-01) from GE Healthcare; Lipofectamine 2000 (11668-019) and Lipofectamine RNAiMAX (13778-150) from Invitrogen; anti-Flag M2 magnetic beads (M8823) and 2-BP (238422) from Sigma-Aldrich; CytoTox 96 Non-Radio cytotoxicity assay kit (G1780) from Promega; Hoechst 33342/PI double stain kit (CA1120) from Solarbio; and FITC annexin V appotosis kit I (556547) from BD Biosciences. .. Cell culture and treatments The human colon cancer cell lines HCT116 wild type and BAK −/− BAX −/− (DKO), the mouse embryonic fibroblasts MEFs, the human ovarian cancer cell lines including Ovcar3, Ovcar5, Ovcar8, A2780, HeyA8, OV90, COV362, SKOV3, Ovise, and the hematoma cell lines including V937, MOLT-3, MOLT-4, THP-1, SKW6.4, H9, Jurkat, K562, KG1a, RL, ML-1, HL-60, HT, and MV4-11 were kind gifts from Scott H. Kaufmann (Mayo Clinic, Rochester, MN).

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  • 93
    Thermo Fisher lipofectamine messengermax
    mRNA delivery into hiPS cells. ( A ) Schematic illustration of EGFP mRNA. ( B ) Fluorescent microscopy images of hiPS cells 24h post transfection with EGFP mRNA in the DEF-CS culture system with <t>Lipofectamine</t> <t>MessengerMAX</t> TM transfection reagent. The upper panels show brightfield images and the lower panels show fluorescent recordings. ( C ) FACS profiles of hiPS cells 24h post transfection or nucleofection with EGFP mRNA. Grey = control cells (non-transfected).
    Lipofectamine Messengermax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine 3000 transfection reagent
    MiR-181d-5p targets KLF6 to ameliorate H/R injury. HK-2 cells were co-transfected with the KLF6 plasmid and miR-181d-5p mimic with <t>Lipofectamine</t> 3000 and, 72 h later, were treated with hypoxia (l% oxygen) for 24 h/reoxygenation for 3 h. (A,B) Quantitative analysis of HIF1-α, KIM-1, and caspase-3 expression in HK-2 cells treated with or without miR-181d-5p and KLF6 ( n = 4 or 5 per group). (C) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 4 per group). The data are presented as the means ± SDs. ∗ P
    Lipofectamine 3000 Transfection Reagent, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 292 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher lipofectamine mix
    Comparison of transduction efficiencies in different cell lines. Blue columns represent the results obtained with the method of mixing <t>lipofectamine</t> 3000 and lipofectamine LTX to transfect HEK-293T cells, concentrating the resulting virus supernatant through Vivaspin 10 000 MW columns, and gently pelleting the recipient cells prior to cell transduction. In comparison, red columns stand for results obtained with a commonly used PEI protocol without concentrating viruses and cells. * P
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    Thermo Fisher mock transfected
    Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of <t>transfected</t> SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p
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    mRNA delivery into hiPS cells. ( A ) Schematic illustration of EGFP mRNA. ( B ) Fluorescent microscopy images of hiPS cells 24h post transfection with EGFP mRNA in the DEF-CS culture system with Lipofectamine MessengerMAX TM transfection reagent. The upper panels show brightfield images and the lower panels show fluorescent recordings. ( C ) FACS profiles of hiPS cells 24h post transfection or nucleofection with EGFP mRNA. Grey = control cells (non-transfected).

    Journal: Genes

    Article Title: Efficient Generation and Correction of Mutations in Human iPS Cells Utilizing mRNAs of CRISPR Base Editors and Prime Editors

    doi: 10.3390/genes11050511

    Figure Lengend Snippet: mRNA delivery into hiPS cells. ( A ) Schematic illustration of EGFP mRNA. ( B ) Fluorescent microscopy images of hiPS cells 24h post transfection with EGFP mRNA in the DEF-CS culture system with Lipofectamine MessengerMAX TM transfection reagent. The upper panels show brightfield images and the lower panels show fluorescent recordings. ( C ) FACS profiles of hiPS cells 24h post transfection or nucleofection with EGFP mRNA. Grey = control cells (non-transfected).

    Article Snippet: For prime editing, 1 μL of Lipofectamine MessengerMAX was added to 25 μL of Opti-MEM medium, vortexed briefly, and incubated for 10 min at RT.

    Techniques: Microscopy, Transfection, FACS

    MiR-181d-5p targets KLF6 to ameliorate H/R injury. HK-2 cells were co-transfected with the KLF6 plasmid and miR-181d-5p mimic with Lipofectamine 3000 and, 72 h later, were treated with hypoxia (l% oxygen) for 24 h/reoxygenation for 3 h. (A,B) Quantitative analysis of HIF1-α, KIM-1, and caspase-3 expression in HK-2 cells treated with or without miR-181d-5p and KLF6 ( n = 4 or 5 per group). (C) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 4 per group). The data are presented as the means ± SDs. ∗ P

    Journal: Frontiers in Physiology

    Article Title: MiR-181d-5p Targets KLF6 to Improve Ischemia/Reperfusion-Induced AKI Through Effects on Renal Function, Apoptosis, and Inflammation

    doi: 10.3389/fphys.2020.00510

    Figure Lengend Snippet: MiR-181d-5p targets KLF6 to ameliorate H/R injury. HK-2 cells were co-transfected with the KLF6 plasmid and miR-181d-5p mimic with Lipofectamine 3000 and, 72 h later, were treated with hypoxia (l% oxygen) for 24 h/reoxygenation for 3 h. (A,B) Quantitative analysis of HIF1-α, KIM-1, and caspase-3 expression in HK-2 cells treated with or without miR-181d-5p and KLF6 ( n = 4 or 5 per group). (C) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 4 per group). The data are presented as the means ± SDs. ∗ P

    Article Snippet: After 24 h, 293T cells were co-transfected with the miR-181d mimic or a scrambled miRNA sequence and PGL3-KLF6-wt or PFL3-KLF6-mut using Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific, Waltham, MA, United States).

    Techniques: Transfection, Plasmid Preparation, Expressing

    KLF6 overexpression exacerbated the hypoxia-induced decline in renal function, renal tubular cell apoptosis, and inflammatory response. HK-2 cells were transfected with KLF6 plasmid and KLF6 shRNA plasmid or scrambled plasmid with Lipofectamine 3000 and, 72 h later, were incubated in normoxia (control) or treated with hypoxia (1% oxygen) for 24 h/reoxygenation for 3 h. (A) KLF6 protein expression in HK-2 cells treated with or without KLF6 ( n = 3 per group). (B,C) qRT-PCR was used to measure miR-181d-5p, KIM-1 and HIF1-α levels after KLF6 transfection ( n = 5 per group). (D) Annexin V-FITC/PI double staining was utilized to evaluate apoptosis after KLF6 transfection. This experiment was repeated three times. (E) KLF6 increased NF-KB expression. HK-2 cells were transfected with or without the KLF6 plasmid. The results shown are from Western blot analysis of NF-KB and I-KB. β-Actin and Lamin-A were used as internal controls for I-KB and NF-KB, respectively ( n = 3 per group). (F) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 3 per group). The data are presented as the means ± SDs. * P

    Journal: Frontiers in Physiology

    Article Title: MiR-181d-5p Targets KLF6 to Improve Ischemia/Reperfusion-Induced AKI Through Effects on Renal Function, Apoptosis, and Inflammation

    doi: 10.3389/fphys.2020.00510

    Figure Lengend Snippet: KLF6 overexpression exacerbated the hypoxia-induced decline in renal function, renal tubular cell apoptosis, and inflammatory response. HK-2 cells were transfected with KLF6 plasmid and KLF6 shRNA plasmid or scrambled plasmid with Lipofectamine 3000 and, 72 h later, were incubated in normoxia (control) or treated with hypoxia (1% oxygen) for 24 h/reoxygenation for 3 h. (A) KLF6 protein expression in HK-2 cells treated with or without KLF6 ( n = 3 per group). (B,C) qRT-PCR was used to measure miR-181d-5p, KIM-1 and HIF1-α levels after KLF6 transfection ( n = 5 per group). (D) Annexin V-FITC/PI double staining was utilized to evaluate apoptosis after KLF6 transfection. This experiment was repeated three times. (E) KLF6 increased NF-KB expression. HK-2 cells were transfected with or without the KLF6 plasmid. The results shown are from Western blot analysis of NF-KB and I-KB. β-Actin and Lamin-A were used as internal controls for I-KB and NF-KB, respectively ( n = 3 per group). (F) ELISAs were used to measure 1L-6 and TNF-α expression levels in the cell supernatant ( n = 3 per group). The data are presented as the means ± SDs. * P

    Article Snippet: After 24 h, 293T cells were co-transfected with the miR-181d mimic or a scrambled miRNA sequence and PGL3-KLF6-wt or PFL3-KLF6-mut using Lipofectamine 3000 transfection reagent (L3000015, Thermo Fisher Scientific, Waltham, MA, United States).

    Techniques: Over Expression, Transfection, Plasmid Preparation, shRNA, Incubation, Expressing, Quantitative RT-PCR, Double Staining, Western Blot

    Comparison of transduction efficiencies in different cell lines. Blue columns represent the results obtained with the method of mixing lipofectamine 3000 and lipofectamine LTX to transfect HEK-293T cells, concentrating the resulting virus supernatant through Vivaspin 10 000 MW columns, and gently pelleting the recipient cells prior to cell transduction. In comparison, red columns stand for results obtained with a commonly used PEI protocol without concentrating viruses and cells. * P

    Journal: Biology Methods & Protocols

    Article Title: Process for an efficient lentiviral cell transduction

    doi: 10.1093/biomethods/bpaa005

    Figure Lengend Snippet: Comparison of transduction efficiencies in different cell lines. Blue columns represent the results obtained with the method of mixing lipofectamine 3000 and lipofectamine LTX to transfect HEK-293T cells, concentrating the resulting virus supernatant through Vivaspin 10 000 MW columns, and gently pelleting the recipient cells prior to cell transduction. In comparison, red columns stand for results obtained with a commonly used PEI protocol without concentrating viruses and cells. * P

    Article Snippet: The lipofectamine mix consisted of 160 µl lipofectamine LTX (Thermo Fisher Scientific), 40 µl lipofectamine 3000 (Thermo Fisher Scientific), and 2.1 ml Opti-MEM medium (Thermo Fisher Scientific).

    Techniques: Transduction

    Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of transfected SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Mitochondria activity, mitochondrial respiratory enzyme, and ATP level of transfected SCs. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Mitochondrial staining in SCs. Scale bar: 50 μm. ( B ) Relative fluorescence intensity for mitochondrial staining. ( C ) NADH level. Activities of ( D ) cytochrome c oxidase and ( E ) ATPase synthase in the mitochondria of SCs. ( F ) ATP level in SCs. ( G ) ATP5A1 mRNA relative level. Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Activity Assay, Transfection, Staining, Fluorescence

    Chicken SC protein expression. ( A ) Representative western blot analysis of protein bands in SCs exposed to 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S5 . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F ) Representative western blot analysis of protein bands in SCs trasfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S6 . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I ) p-mTOR/mTOR in transfected SCs. One independent replicate on western blot analysis of protein bands in SCs is presented in Supplementary Figure S7 . Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Chicken SC protein expression. ( A ) Representative western blot analysis of protein bands in SCs exposed to 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S5 . Relative protein levels of ( B ) NRF2, KEAP1, PRDX4, ( C ) ATP5A, ( D ) p-AMPKα/AMPKα, and ( E ) p-mTOR/mTOR in SCs exposed to plasma. ( F ) Representative western blot analysis of protein bands in SCs trasfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. Uncropped immunoblot scans are presented in Supplementary Figure S6 . Relative protein levels of ( G ) ATP5A, ( H ) p-AMPKα/AMPKα, and ( I ) p-mTOR/mTOR in transfected SCs. One independent replicate on western blot analysis of protein bands in SCs is presented in Supplementary Figure S7 . Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Expressing, Western Blot, Transfection

    Expression of miR-7450 and mRNA levels of AMPKα and mTOR in SCs. ( A ) miR-7450 relative level and ( B ) relative mRNA levels of AMPKα and mTOR in SCs exposed to 22.0 kV of plasma for 120 s. ( C ) miR-7450 relative level and ( D ) relative mRNA levels of AMPKα and mTOR in SCs transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. RT-PCR analysis of a non-target gene ( POU1F1 ) and an unrelated target gene ( PDE10A ) of miR-7450 in transfected SCs is presented in Supplementary Figure S4 . Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: Expression of miR-7450 and mRNA levels of AMPKα and mTOR in SCs. ( A ) miR-7450 relative level and ( B ) relative mRNA levels of AMPKα and mTOR in SCs exposed to 22.0 kV of plasma for 120 s. ( C ) miR-7450 relative level and ( D ) relative mRNA levels of AMPKα and mTOR in SCs transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. RT-PCR analysis of a non-target gene ( POU1F1 ) and an unrelated target gene ( PDE10A ) of miR-7450 in transfected SCs is presented in Supplementary Figure S4 . Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Expressing, Transfection, Reverse Transcription Polymerase Chain Reaction

    SC viability and apoptosis after miRNA transfection. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Relative viability of SCs. ( B ) Flow cytometric analysis of SC cell apoptosis. ( C ) JC-1 staining of SCs. Scale bar: 50 μm. ( D ) Relative green/red fluorescence intensity for JC-1 staining. Data are represented as the mean ± SD (n = 3 per group). * p

    Journal: Scientific Reports

    Article Title: MicroRNA-7450 regulates non-thermal plasma-induced chicken Sertoli cell apoptosis via adenosine monophosphate-activated protein kinase activation

    doi: 10.1038/s41598-018-27123-8

    Figure Lengend Snippet: SC viability and apoptosis after miRNA transfection. Chicken SCs were transfected with miR-7450 agomir and antagomir, and miR-7450 agomir-transfected group treated with 22.0 kV of plasma for 120 s. ( A ) Relative viability of SCs. ( B ) Flow cytometric analysis of SC cell apoptosis. ( C ) JC-1 staining of SCs. Scale bar: 50 μm. ( D ) Relative green/red fluorescence intensity for JC-1 staining. Data are represented as the mean ± SD (n = 3 per group). * p

    Article Snippet: At 60–80% confluence, SCs were mock-transfected (Lipofectamine® RNAiMAX Regent only; Thermo Fisher Scientific) or transfected with complexes of Lipofectamine® RNAiMAX, miR-7450 agomir negative control (NC; 50 nM), miR-7450 antagomir NC (100 nM), miR-7450 agomir (chemically-modified double-stranded miRNA mimic; 50 nM) in the presence or absence of non-thermal plasma treatment at 22.0 kV for 120 s, and miR-7450 antagomir (chemically-modified single-stranded miRNA inhibitor; 100 nM) from GenePharma Co., Ltd. (Shanghai, China), according to the manufacturer’s protocol.

    Techniques: Transfection, Flow Cytometry, Staining, Fluorescence